Ledgf peptides and formulations thereof for treatment of degenerative disorders

ABSTRACT

LEDGF peptides with anti-protein aggregation activity and methods of use are provided. The LEDGF peptides disclosed herein demonstrate an ability to treat degenerative diseases and diseases with various cellular stresses including oxidative stress and protein-aggregation stress. In addition, extended release formulations, including formulations suitable for ophthalmic administration are provided.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/649,847 filed May 21, 2012, and International Patent ApplicationNo. PCT/US2012/065620 filed on Nov. 16, 2012. The contents of theabove-identified priority applications are hereby fully incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates generally to novel peptides of lensepithelium derived grown factor (LEDGF) and compositions thereof for usein treating degenerative diseases and diseases with various cellularstresses including oxidative stress and protein-aggregation stress. Morespecifically, the present inventions relates to novel formulations ofLEDGF₁₋₃₂₆ with enhanced stability and sustained delivery profiles andtheir use in treating protein aggregation-mediated diseases, age-relateddiseases, and degenerative diseases.

BACKGROUND

Diseases of the posterior segment of eye which includes age relatedmacular degeneration (AMD) and retinitis pigmentosa (RP) are the leadingcause of blindness in United States. (Jager et al., N ENGL J MED, 2008.358(24): 2606-17). Currently, about 8 million individuals are sufferingfrom AMD in the United States and by 2020 this number is expected toreach 12 million. (Jager et al.; Friedman et al. Arch Ophthalmol, 2004.122(4): p. 564-72). Dry form of AMD (Dry AMD) associated with chronicoxidative stress and inflammation accounts for 90% of AMD cases. (Libbyet al. Adv Exp Med Biol, 2010. 664: p. 403-9; Stuen. Generations, 2003.27: p. 8-14). On the other hand RP is a genetically inherited diseasecaused by more than 50 different gene mutations. (Ohguro, H., et al.,Nihon Ganka Gakkai Zasshi, 2002. 106(8): p. 461-73; Dryja, et al.,Nature, 1990. 343(6256): p. 364-6.) Around 1.5 million people worldwidecurrently suffer from RP. (Hartong, et al., Lancet, 2006. 368(9549): p.1795-809).

The unique anatomy and physiology of the eye is a major hurdle in theadvancement of drug therapeutic for the back of the eye includingretinal degenerative diseases. (Kompella et al., Ther Deliv. 1(3): p.435-56). Topical routes of administration are inefficient in deliveringdrugs to the back of the eye because of the presence of various staticbarriers (cornea, conjunctiva, and sclera among others tissues) anddynamic barriers (blinking, tear film, tear turn over, and inducedlacrymation). (Gaudana, R., et al., Ocular drug delivery. Aaps J, 2010.12(3): p. 348-60; Thrimawithana et al. Drug Discov Today, 2011. 16(5-6):p. 270-7). On the other hand blood retinal barrier (BRB), systemicdegradation, systemic side effects, and low concentrations at targetsite are major challenges for the intravenous route. Other routes suchas intra cameral, periocular, subretinal have their own subset ofproblems sharing some issues in common with topical and systemic routeof administration (Baid et al. Drug Development and the back of the eye.ed. Kompella UB. 2010, p. 409-448: Springer). Local delivery such as anintravitreal injection places the drug to close proximity to the retina(the target tissue for retinal degenerative diseases) and thus is themost effective route in delivering drug to retina. However, frequentintravitreal injections of the drug leads to various complications suchas retinal detachment, retinal hemorrhage, endopthalmitis, increasedintraocular pressure, and not to mention patient compliance andinfections. (Peyman et al. Retina, 2009. 29(7): p. 875-912; Wu et al.Semin Ophthalmol, 2009. 24(2): p. 100-5). Thus there is a need of forcompositions and delivery system that can extend the retention of drugsin the eye.

Novel drug delivery systems have gained major attention which couldsustain or control the release of drug for extended period of time aswell as increase the stability and bioavailability of therapeutic agentssuch as proteins, genes and other small molecules. Biodegradable (PLGA,PCL) and non-biodegradable (e.g Vitraset and Retisert) implants providesa platform for sustaining release of drug over several months to years.However, erratic drug release profile for biodegradable implants andrequirement of highly invasive eye surgery are few drawbacks. Micro andnanoparticles provide sustained release of encapsulated molecules forweeks to months. However, use of organic solvents such asdichloromethane during preparation denatures and reduces proteinefficacy leading to non-effective treatment. Further encapsulationefficiency, controlled particles size, and sterility during preparationsare among the other hurdles. Iontophoresis, microneedles, ultrasoundbased ocular deliver have also been tried, however, the major advancesare with the small molecule drugs and still are in investigation stageand needs validations to establish their efficacy and safety. Thus non-or minimally-invasive, controlled, and sustained delivery to theposterior segment is becoming extremely vital with escalating advancesin the emerging therapies for retinal degenerations.

SUMMARY

The present invention is directed to biologically active peptides ofLEDGF that can be produced in high quantity, purity, or both. Forexample, the present invention is directed to peptides of LEDGF that canbe produced at, or greater than, 20 mg per liter of culture and at, orgreater than, 90% purity as quantified by SDS-PAGE and SEC-HPLC. In oneexemplary embodiment the peptide is approximately a 40 kDa monomer, thatmay exist as an 80 kDa dimer. In another exemplary embodiment, thepeptide has primarily a random coil structure and includes an N-terminalstress related binding domain, and optionally a TAT binding domain.

In another exemplary embodiment, the peptide comprises amino acids 1-326of LEDGF (LEDGF₁₋₃₂₆). In yet another exemplary embodiment, the peptidecomprises SEQ ID NO: 2. In another exemplary embodiment, the peptidecomprises an amino acid sequence with at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% sequence identitywith SEQ ID NO: 2. In addition, the present invention includes nucleicacid sequences encoding SEQ ID NO: 2, or nucleic acid sequences encodingamino acid sequences having at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% sequence identity with SEQ IDNO: 2. The present invention further includes vectors containing suchnucleic acid sequences. In one exemplary embodiment, the vector is apET-28a(+) vector.

In another aspect, the present invention comprises compositionscontaining the LEDGF peptides. In one exemplary embodiment, thecomposition comprises the LEDGF peptide in combination with apharmaceutical carrier, diluent, excipient, or combination thereof. Inanother exemplary embodiment, the composition comprises the LEDGFpeptide associated with or bound to colloidal metal particles, such aszinc, to form nano-assemblies. In yet another exemplary embodiment, thecompositions comprise the LEDGF peptide encapsulated or bound to aninner particle loaded into a porous outer particle. In certain exemplaryembodiments, the LEDGF peptide used in the above compositions isLEDGF₁₋₃₂₆.

In another aspect, the present invention is directed to methods oftreating protein aggregation-mediated diseases by administering theabove LEDGF peptide compositions to a patient in need thereof. Incertain exemplary embodiments, the protein aggregation-mediated diseaseis a retinal degeneration disease. Exemplary retinal degenerationdiseases include, but are not limited to, age related maculardegeneration (AMD) retinitis pigmentosa (RP) and diabetic retinopathy(DR). In another exemplary embodiment, the protein aggregation-mediateddiseases are neurodegenerative diseases including, but not limited to,Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease(HD), amyotrophic lateral sclerosis, or a prion disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an immunoblot of purified full-length LEDGF indicating thatattempts to purify full length LEDGF result in an unstable andfragmented product.

FIG. 2 is a diagram comparing full length LEDGF to LEDGF₁₋₃₂₆.

FIG. 3 is a picture of an agarose gel showing the successful cloning ofa nucleic acid sequence encoding LEDGF₁₋₃₂₆ into pET-28 a (+).Ledgf₁₋₃₂₆ gene was PCR amplified and then ligated into pET-28 a (+)vector. Lane 1: PCR amplified Ledgf₁₋₃₂₆ gene, Lane 2: uncut circularpET-28 a (+), Lane 3: linearized BamHI digested pET-28 a (+), Lane 4:uncut circular pLEDGF₁₋₃₂₆ (pET-28 a (+) ligated with Ledgf₁₋₃₂₆), Lane5: linearized BamH1 digested pLEDGF₁₋₃₂₆, Lane 6: linearized HindIIIdigested pLEDGF₁₋₃₂₆, Lane 7: BamHI and HindIII double digestedpLEDGF₁₋₃₂₆, Lane 8: PCR amplification of Ledgf₁₋₃₂₆ gene frompLEDGF₁₋₃₂₆.

FIGS. 4A-C are set of graphs showing successful expression andpurification of LEDGF₁₋₃₂₆: A) SDS-PAGE—Lane 1 Protein marker(Fermentas, Glen Burnie, Md.), Lane 2 uninduced cell lysate, Lane 3 IPTGinduced cell lysate, Lane 4 Soluble fraction of lysate, Lane 5LEDGF₁₋₃₂₆ obtained from FPLC-cation exchange column, Lane 6 LEDGF₁₋₃₂₆obtained from FPLC-gel filtration column; B) FPLC-cation exchangechromatogram; C) FPLC-gel filtration chromatogram.

FIGS. 5A-D are set of graphs showing certain physical characteristics ofLEDGF₁₋₃₂₆. A) SEC-HPLC: LEDGF₁₋₃₂₆ was size separated on AgilentBio-SEC column using 25 mM Tris-HCL buffer containing 1 mM CaCl₂, pH7.0. LEDGF₁₋₃₂₆ was eluted primarily as single peak at 11.5 min withabout 5% higher molecular weight species. B) MALDI-TOF: LEDGF₁₋₃₂₆ has amolecular weight of 40 kDa, and may exist as a dimer. C) DLS: LEDGF₁₋₃₂₆size was analyzed in 25 mM phosphate buffer, pH 7.0 using Nano ZS.LEDGF₁₋₃₂₆ had a monodispersed population of about 10 nm diameter, withthe absence of aggregates. D) SDS-PAGE: LEDGF₁₋₃₂₆ was size separated ona 4-15% SDS-PAGE gel under reducing (beta-mercaptoethanol and boiling)and non-reducing (no beta-mercaptoethanol and no boiling) conditions.The non-reducing gel indicated the presence of dimers of LEDGF₁₋₃₂₆.

FIG. 6A-B are a set of graphs representing additional data on predictedstructural features of LEDGF₁₋₃₂₆. A) Circular dichroism: Secondarystructures of 500 μg/ml LEDGF₁₋₃₂₆ in 25 mM phosphate buffer pH 7.0 wasanalyzed using Chirascan. No characteristic peaks were obtained forα-helix or β-sheets. Further, presence of the strong negative signal at200 nm indicated that LEDGF₁₋₃₂₆ is primarily a random coiled protein.B) LEDGF₁₋₃₂₆ 3-D Model: LEDGF₁₋₃₂₆ 3-D structure was predicted byI-Tasser protein modeling server based on its amino acid sequence andhomology with proteins whose structures are available in protein databank. According to the 3-D model, LEDGF₁₋₃₂₆ is a random coiled protein.

FIG. 7A-F provides additional graphs representing additional data on thepredicted structural features of LEDGF₁₋₃₂₆. Fluorescencespectroscopy-chemical denaturation: A) Fluorescence spectra of 300 μg/mlof LEDGF₁₋₃₂₆ incubated with 0-6 M urea in 25 mM phosphate buffer at pH7.0 was recorded from 300 to 400 nm at the excitation wavelength of 280nm. B) Ratio of fluorescence intensity at 340/356 nm was plotted againstthe urea concentration to determine conformational stability parameters.C) CD spectra of 300 μg/ml of LEDGF₁₋₃₂₆ incubated with 0-6 M urea in 25mM phosphate buffer at pH 7.0 was recorded from 220 to 260 nm. D) CDsignal at 230 nm was plotted against the urea concentration to determineconformational stability parameters. E) LEDGF₁₋₃₂₆ was denatured usingheat and the corresponding changes in the CD signal were recorded from215 to 250 nm. F) CD signal at 222 nm was plotted against thetemperature to determine the melting temperature of LEDGF₁₋₃₂₆.

FIG. 8A-B is a set of graphs showing the ability of LEDGF₁₋₃₂₆ to rescueARPE-19 cells from aggregation mediated stress. ARPE-19 cells weretreated with LEDGF₁₋₃₂₆ for 48 hours in the A) absence or B) presence ofaggregation stress.

FIGS. 9A-B is a set of graphs demonstrating that LEDGF₁₋₃₂₆ delays thefunctional loss of photoreceptors in RCS rats. A) Scotopic ERG wasrecoded using 0.4 log cd-s/m² flashes and scotopic B-wave amplitude wasplotted against the age of rats. For photopic ERG, rats were lightadapted using 30 cd/m² background light for 3 min before recording theERG. B) Photopic B-wave amplitude was plotted against the age of rats.Three ERGs were averaged for individual rat and then b-wave amplitudewithin each group was averaged to get the mean. Data represent mean±S.D.for N=3. *, p<0.05 compared to corresponding untreated group.

FIG. 10 is a set of graphs showing that LEDGF₁₋₃₂₆/zinc nano-assembliesare stable after dilution.

FIG. 11 A-C is a set of graphs showing LEDGF1-326 tertiary structure,secondary structure and size changes in absence or presence of additives

FIG. 12 is a picture of a SDS-PAGE gel of LEDGF₁₋₃₂₆ loaded underreducing conditions.

FIGS. 13A-B is a set of graphs showing quantities of soluble protein andinsoluble aggregates of LEDGF₁₋₃₂₆ in the presence and absence ofvarious additives.

FIG. 13C is a picture of microcentrifuge tubes showing absence ofinsoluble aggregates of LEDGF₁₋₃₂₆ in the presence of various additives.

FIG. 14 are graphs depicting the immunoreactivity of LEDGF₁₋₃₂₆ in thepresence and absence of various additives.

FIG. 15A-C is a set of graphs showing the biophysical characterizationof structural integrity and conformational stability of LEDGF₁₋₃₂₆ inthe presence of additives. A) Fluorescence intensity of LEDGF₁₋₃₂₆ at342 nm with excitation at 280 nm as function of time in the presence ofvarious additives. B) Circular dichroism (CD) of LEDGF₁₋₃₂₆ at 208 nm asa function of time and additives. C) Hydrodynamic size of LEDGF₁₋₃₂₆ asa function of time and additives.

FIG. 16 is a picture of a SDS-PAGE gel providing a molecular weightanalysis of LEDGF₁₋₃₂₆ as a function of time.

FIGS. 17A-D is a set of graphs showing formation of LEDGF₁₋₃₂₆nanoassemblies in the presence of zinc. A) Dynamic light scattering.LEDGF₁₋₃₂₆ forms nanoassemblies in the presence of zinc. B) Circulardichorism. The secondary structure of LEDGF₁₋₃₂₆ is altered in thepresence of zinc. C) Fluorescence spectroscopy. LEDGF₁₋₃₂₆ fluorecencespectra is quenched in the presence of zinc indicating the exposture ofhydrophobic residues to more polar environment. D) Ultra violetspectroscopy: LEDGF₁₋₃₂₆ uv absorbance was quenched in the presence of0.1 mM and 1 mM zinc but not with 10 mM zinc.

FIG. 18 is a set of TEM images of LEDGF₁₋₃₂₆ zinc nanoassemblies.

FIGS. 19A-D is a set of graphs demonstrating the reversible formation ofLEDGF₁₋₃₂₆ nanoassemblies.

FIGS. 20A-D is a set of graphs and an image of an SDS-PAGE geldemonstrating the increased stability of LEDGF₁₋₃₂₆ nanoassemblies overtime.

FIG. 21 is a graph demonstrating increased uptake of LEDGF₁₋₃₂₆nanoassemblies by ARPE-19 cells.

FIG. 22 is a graph depicting the results of a MTT assay demonstratingthe ability of LEDGF₁₋₃₂₆ nanoassemblies to rescue ARPE-19 cells fromserum starvation.

FIGS. 23A-D is a set of graphs demonstrating the ability of LEDGF₁₋₃₂₆nanoassemblies to reduce retinal degeneration as examined usingelectroretinography.

FIGS. 24A-H is a set of graphs demonstrating the persistence ofLEDGF₁₋₃₂₆ nanoassemblies in vitreous for at least 14 days as measuredby detecting the fluorescence signal of Alexa-LEDGF₁₋₃₂₆ in normal SDrats. A) Demonstrates the results of a blank scan before intravitrealinjection of the SD rat eye. B) and C) demonstrate the standard curvefor control and LEDGF₁₋₃₂₆ assemblies respectively. D) and E)demonstrate the fluorescence signal in various tissues includingvitreous, choroid-RPE, and aqueous humor as obtained from the Flurotronscans; F), G), and H) convert the fluorescent signal measured above toactual LEDGF₁₋₃₂₆ nanoassemblies for vitreous, choroid-RPF, and aqueoushumor respectively.

FIG. 25 is a graph demonstrating the ability of nanoassemblies topreserve the immunoreactivity of LEDGF₁₋₃₂₆.

FIGS. 26A-B are A) a set of images of a cell count assay of ARPE-19cells transfected with LEDGF₁₋₃₂₆ and B) graphs demonstrating theresults of the cell count assay as a function of time and concentrationof LEDGF₁₋₃₂₆.

FIG. 27 is a graph demonstrating the results of an assay measuringphagocytic activity and indicating increased phagocytic activity inARPE-19 cells transfected with LEDGF₁₋₃₂₆.

FIGS. 28A-D are a set of images and graphs demonstrating the results ofa histological analysis of SD rat eyes injected with LEDGF₁₋₃₂₆. A) is agraphical depiction of a vertical section of an eye at the site ofintravitreal injection. B) is a set of images of cross sections ofnormal SD, untreated RCS, and LEDGF₁₋₃₂₆ treated RCS retinas. C) and D)are graphs depicting the measured thickness of outer and inner nuclearlayers of the eye in control and LEDGF₁₋₃₂₆ treated retinasrespectively.

FIG. 29 is panel of immunofluorescence images of control and LEDGF₁₋₃₂₆treated rat SD retinas.

FIG. 30 is a graph showing the cumulative release of His-LEDGF₁₋₃₂₆ fromexample PinP compositions

FIG. 31 are graphs showing the results of non-invasive ocularfluorophotometry after intravitreal injection in rat eyes withAlexa-His-LEDGF₁₋₃₂₆.

DETAILED DESCRIPTION Definitions

As used herein “retina and retinal” refers both to the retina as well asthe general posterior segment of the eye adjacent to the retina.

As used herein “treating or treatment” refers to a complete reversal orelimination of the underlying disease, a temporary or sustainedprevention of disease progression, a temporary or sustained regressionof the disease, and amelioration of one or more symptoms associated withthe disease.

The terms “peptide,” “polypeptide” and “protein” are usedinterchangeably herein. Unless otherwise noted, the terms refer to apolymer having at least two amino acids linked through peptide bounds.The terms thus include oligopeptides, protein fragments, analogs,derivatives, glycosylated derivatives, pegylated derivatives, fusionproteins and the like.

As used herein, “sequence identity/similarity” refers to the identityof, or similarity of two or more amino acid sequences. Sequence identitycan be measured in terms of percentage identity, the higher thepercentage, the more identical the sequences are. Methods of alignmentand sequences for comparison are well known in the art. Various programsand alignment algorithms are described in: Smith & Waterman, Adv. Appl.Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443; Pearson &Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene,73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al.Nuc. Acids. Res. 16:10881-10, 1990, present detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Research Tool (BLAST) (Altschule et l al.J. Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn, and tblastx. Blastp is used tocompare amino acid sequences. Additional information can be found at theNCBI web site.

In general, once aligned, the number of matches is determined bycounting the number of positions where an identical amino acid residueis present in both sequences. The percent identity is determined bydividing the number of matches either by the length of the sequence setforth in the identified sequences, or by an articulated length (such as100 consecutive nucleotides or amino acids residues from a sequence setforth in an identified sequence), followed by multiplying the resultingvalue by 100.

INTRODUCTION

Full length LEDGF has the ability to rescue retinal pigment epithelialcells from P23H mutant rhodopsin aggregation induced stress (Baid etal., PLoS One. 6(9): p. e24616). However, the use of therapeuticpeptides is challenging due to the need of the protein to maintain aspecific three-dimensional structure in order to remain biologicallyactive. In addition, production and biosynthesis often fail because of alack of protein stability throughout the biosynthesis and purificationprocess. Further, in order to use protein therapeutics effectively, itis often necessary to achieve production of several milligrams in orderto characterize the proteins properties effectively. For example, fulllength LEDGF yields only 0.9 mg per 500 ml, well short of the tens ofmilligrams needed to properly characterize the protein, and results in afragmented product.

The present invention provides peptides of LEDGF that maintain thefull-length protein's cell surviving activity while allowing productionand purification of LEDGF peptides in high quantity and purity. Further,the LEDGF peptides of the present invention have anti-proteinaggregation activity. The LEDGF peptides of the present inventiondemonstrate an ability to treat degenerative diseases and diseasesassociated with various cellular stresses including oxidative stress andprotein-aggregation stress. Accordingly, a molecule like the LEDGFpeptides of the present invention may represent a universal therapeuticprotein for treating multiple protein-aggregation mediated diseases,including other retinal degenerative and neurodegenerative diseases. Thepresent invention further comprises extended release formulations of theLEDGF peptides useful in treating the above diseases.

LEDGF Peptides

The LEDGF peptides of the present invention contain N-terminal peptidesof full-length LEDGF. In one exemplary embodiment, the LEDGF peptidecomprises the LEDGF N-terminal stress related binding domain. While notlimited by the following theory, LEDGF's ability to function as atranscription factor and initiate transcription of other stress responsegenes may contribute to LEDGF peptide's ability to protect againstprotein aggregation-mediated diseases. Alternatively, LEDGF peptides maybind to mis-folded proteins, either directly or through otherintermediary proteins, and facilitate normal folding, or ubiquitnationof mis-folded proteins to ensure proteolytic degradation. In certainexemplary embodiments, the LEDGF peptide may further comprise a TATbinding domain.

In one exemplary embodiment, the LEDGF peptide is LEDGF₁₋₃₂₆ (SEQ ID NO:2). LEDGF₁₋₃₂₆ was purified to near homogeneity. LEDGF₁₋₃₂₆ has aprimarily random coiled structure, is stable at room temperature, andexists as a 40 kDa monomer and/or 80 kDa dimer. As described in furtherdetail in the Examples section below, LEDGF₁₋₃₂₆ was able to preventP23H mutant rhodopsin mediated aggregation stress in ARPE-19 cells.Single intravitreal injection of LEDGF₁₋₃₂₆ reduced the functional lossof photoreceptors in retinal degenerative rat model for over eightweeks.

LEDGF peptides of the present invention also include LEDGF peptideshaving at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, or at least about 95% sequenceidentity with LEDGF₁₋₃₂₆. In one exemplary embodiment, LEDGF peptidesinclude peptides encompassing the N-terminal stress related bindingdomain of full-length LEDGF and at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, or at leastabout 95% sequence identity with LEDGF₁₋₃₂₆. In another exemplaryembodiment, LEDGF peptides include peptides with the N-terminal stressrelated binding domain and a TAT binding domain and at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, or at least about 95% sequence identity with LEDGF₁₋₃₂₆.Further, the present invention includes peptides encompassing more thanor less than the 326 amino acids of SEQ ID NO: 2, wherein the larger orsmaller peptides do not result in a significant decrease in LEDGFbiological activity or stability during biosynthesis and purification.The suitability of an LEDGF peptide for use with the present inventioncan be determined by one of ordinary skill in the art by assessing theputative peptide's similarity to the biophysical and biochemicalproperties and biological activity using the assays described in theExamples section below. One of ordinary skill can predictably recognizethat LEDGF peptides with similar biophysical properties, biochemicalproperties, and biological activity to LEDGF₁₋₃₂₆ will have similarutility and accordingly fall within the scope of the present invention.

In certain example embodiments, the LEDGF peptides described above aremade synthetically. In certain other example embodiments, the LEDGFpeptides described above are made recombinantly. Host suitable forexpression of the LEDGF peptides include, but are not limited to, E.coli, Saccharomces, Picchia, Bacillus, CHO, BHK, COS, and NSO cells.

Standard Pharmaceutical Formulations

The LEDGF peptides described herein can be provided as physiologicallyacceptable formulations using known techniques. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 19th Edition (1995), describes compositions and formulationssuitable for pharmaceutical delivery of LEDGF peptides disclosed herein.

The formulations in accordance with the present invention can beadministered in the form of a tablet, a capsule, a lozenge, a cachet, asolution, a suspension, an emulsion, a powder, an aerosol, asuppository, a spray, a pastille, an ointment, a cream, a paste, a foam,a gel, a tampon, a pessary, a granule, a bolus, a mouthwash, an implant,in a device, as an eye drop or a transdermal patch.

The formulations include those suitable for oral, rectal, nasal,inhalation, topical (including dermal, transdermal, buccal, and eyedrops), vaginal, parenteral (including subcutaneous, intramuscular,intravenous, intradermal, intraocular, intratracheal, and epidural),ophthalmic (periocular, intraocular, including suprachoroidal,subretinal, and intravitreal), or inhalation administration. In oneexemplary embodiment, the peptides of the present invention areformulated for transcleral, suprachoroidal, subretinal, or intravitrealdelivery. Transcleral delivery includes subconjunctival, subtenons', andretrobulbar transcleral delivery. The formulations can conveniently bepresented in unit dosage form and can be prepared by conventionalpharmaceutical techniques. Such techniques include the step of bringinginto association the active ingredient and a pharmaceutical carrier(s)or excipient(s). In general, the formulations are prepared by uniformlyand intimately bringing into association the active ingredient withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored base, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert base such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels, pastes, and eye drops comprisingthe ingredient to be administered in a pharmaceutical acceptablecarrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken; i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining, in addition to the active ingredient, ingredients such ascarriers as are known in the art to be appropriate.

Formulation suitable for inhalation may be presented as mists, dusts,powders or spray formulations containing, in addition to the activeingredient, ingredients such as carriers as are known in the art to beappropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents; gels; and surgically placed implants.

Nanoassembly Formulations

In certain exemplary embodiments, the LEDGF peptides of the presentinvention may be delivered as nanoparticle assemblies by binding orotherwise associating the LEDGF peptides with colloidal metal particles.Any colloidal metal can be used in the present invention. Colloidalmetals include any water-insoluble metal particle, metallic compounddispersed in liquid water, a hydrosol, or a metal sol. The colloidalmetal particle may be selected from the metals in groups IA, IB, IIB,and IIIB of the periodic table, as well as the transition metals,especially those of group VIII. Exemplary metals include zinc, gold,silver, aluminum, ruthenium, iron, nickel, and calcium. Other suitablemetals include the following in all of their various oxidation states;lithium, sodium, magnesium, potassium, scandium, titanium, vanadium,chromium, manganese, cobalt, copper, gallium, strontium, niobium,molybdenum, palladium, indium, tin, tungsten, rhenium, platinum, andgadolinium. The metals are preferably derived from the appropriate metalcompound in ionic form, for example Al³⁺, Ru³⁺, Zn²⁺, Fe³⁺, Ni²⁺, andCa²⁺.

In one exemplary embodiment, the nanoparticles are formed by adding thecolloidal metal directly to a solution containing the LEDGF peptide. Asused herein “nanoparticles” refer to one or more peptides bound oradsorbed to the surface of a single colloidal metal particle, or apeptide bound to or adsorbed to multiple colloidal metal particles. Byway of example, LEDGF/Zn nanoparticles are formed by adding Zn(II) in acontrolled manner of 10 mM at room temperature. Thereafter thenanoassemblies are allowed to form over 24 hours time period at 37° C.Conditions for formation of other nanoassemblies using other colloidalmetals may be readily determined by one of ordinary skill in the art.

Particle-in-Particle Formulations

In another exemplary embodiment, the LEDGF peptides are formulated asparticle-in-particle extended release formulations. The extended releasecompositions of the present invention comprise an inner particlecontained within a larger porous outer particle, including variousarchitectures such as a nanoparticle in porous microparticle (NPinPMP),small nanoparticle in porous large nanoparticle (SNPinPLNP), and smallmicroparticle in porous large microparticle (SMPinPLMP). The innerparticle is smaller and relatively non-expandable as compared to thelarger particle. The outer particle is expandable and forms asignificantly porous structure during processing that allows theembedding of the inner particle within the outer particle′ porousstructure.

As used in the context of the present invention, a particle isconsidered to expand in the presence of a supercritical fluid if theparticle's initial surface area increases within a range ofapproximately 1.25 to approximately 100 times. In certain exemplaryembodiments, the particle is considered to expand if the particle'sinitial surface area expands within a range of approximately 1.25 toapproximately 5 times, approximately 5 to approximately 25 times,approximately 25 to approximately 50 times, approximately 50 toapproximately 75 times, or approximately 75 to 100 times. Alternatively,a particle is considered to expand if the particle's initial sizeincreases by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50%.

Inner particles of the present invention are made using polymeric ornon-polymeric materials that do not expand in the presence of asupercritical fluid. In certain exemplary embodiments, the nanoparticlematerial is a polymeric material that will not expand in the presence ofsupercritical fluids. In certain exemplary embodiments, the polymericmaterial is a material that will not expand in the presence ofsupercritical carbon dioxide. Examples of suitable polymeric andnon-polymeric materials that may be used in the present inventioninclude polylactide (PLA), poly(glycolic acid), co-polymers of lacticand glycolic acid (PLGA), cellulose derivatives, chitosan, polyethylene(PE), polypropylene, poly(tetrafluoroethylene), poly(ethyleneterephathalate), iron oxide, cerium oxide, zinc oxide, gold, silver,other biocompatible metals and crystals, and silica. Crystallinematerials or those with large crystalline regions are less likely toexpand during supercritical fluid processing. Polymeric inner particlesmay be prepared using conventional emulsion-solvent evaporation methodsor other similarly suitable synthesis method. LEDGF peptides may beencapsulated in the inner particles during formation or loaded on thesurface after formation of the inner particles.

Outer particles of the present invention are made using materials thatexpand in the presence of a supercritical fluid. In certain exemplaryembodiments, the microparticle material is a polymeric material thatexpands in the presence of a supercritical fluid. In certain exemplaryembodiments, the material that expands in the presence of supercriticalcarbon dioxide. Examples of suitable polymeric materials that may beused in the present invention include lactide-co-glycolide, polyamides,polycarbonates, polyakylene glycols, polyalkylene oxides, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, polyvinylpyrrolidone,polyglycolides, and co-polymers thereof. In addition, suitable polymermaterials also include alkyl cellulose, hydroxyalkyl celluloses,cellulose eethers, cellulose esters, nitro celluloses, polymers ofacrylic and methacrylic esters, methyl cellulose, ethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutylmethyl cellulose, cellulose acetate cellulose acetate butyrate,cellulose acetate phthalate, carboxylethyl cellulose, cellulosepoly(methyl methacrylate), poly(elthylmethacrylate),poly(butymethacrylate), poly(vinyl alcohols), poly(vinyl acetate), andpolyvinylpryrrolidone. In general, amorphous materials or those withlarge amorphous regions are suitable for expansion during supercriticalfluid processing. Polymeric outer particles may be prepared usingconventional emulsion-solvent evaporation, or other similarly suitablesynthesis method. In certain exemplary embodiments, LEDGF peptides maybe encapsulated in the outer particles during formation or loaded on thesurface after formation of the outer particles.

The process of generating various particle architectures is achievedusing supercritical fluid flow technology. The resulting organicsolvent-free loading is especially well suited to drugs, such as peptideand nucleotide based drugs, which are susceptible to aggregation ordegradation. For example, LEDGF peptide may be loaded on the surface ofthe inner particle, the outer particle or both; in the matrix of theinner particle, outer particle or both; present in the pores of theouter particle; or a combination thereof. In certain exemplaryembodiments, LEDGF peptides may be present on the surface of the innerparticle. In another exemplary embodiment, LEDGF peptide may be presenton the surface of the inner and outer particle. In yet another exemplaryembodiment, LEDGF peptides may be present in the matrix of the innerparticle. In another exemplary embodiment, LEDGF peptides may be presentin the matrix of both the inner and outer particle. In another exemplaryembodiment, a therapeutic agent may further be present in the porousstructure of the outer particle.

Inner and outer particles are admixed together and exposed to asupercritical fluid under high pressure. In certain exemplaryembodiments, the supercritical fluid is carbon dioxide. Upon exposure tothe supercritical fluid the outer particles expand to create a porousstructure on the outer surface. The supercritical fluid then infuses theinner particles into the outer particles to form particle-in-particleextended release formulations. In one exemplary embodiment, theparticle-in-particle extended release formulations comprise theincorporation of inner nanoparticles having a diameter of approximately1 nm to approximately 900 nm in an outer microparticle having a diameterof approximately 1 μm to approximately 100 μm. In another exemplaryembodiment, the particle-in-particle extended release formulationscomprise the incorporation of an inner nanoparticle having a diameter ofapproximately 1 nm to approximately 300 nm in an outer nanoparticlehaving a diameter of approximately 10 nm to approximately 999 nm. In yetanother exemplary embodiment, the particle-in-particle extended releaseformulations include the incorporation of an inner microparticle havinga diameter of approximately 1 μm to approximately 100 μm in an outermicroparticle having a diameter of approximately 2 μm to approximately500 μm. Selection of an appropriate sized inner and outer particle willdepend on the type of material comprising the particles, the expansiveability of the outer particle in the supercritical fluid used, and thesize of inner particles to be incorporated within the outer particle.These are all factors that can be readily selected for by one ofordinary skill in the art. In general, the size ratio between the innerand outer particle may vary from approximately 1:5 to approximately1:100. In one exemplary embodiment the size ratio may be 1:5, 1:10,1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70,1:75, 1:80, 1:85, 1:90, 1:95, or 1:100

Formation of NPinPMPs may be achieved by exposure of the nanoparticlesand microparticles at approximately 1000 psi to approximately 1400 psi.The time of exposure may vary, for example, from approximately 5 minutesto approximately 2 hours. The temperature may range from 30° C. to 45°C. The selection of an appropriate pressure and temperature range aredetermined primarily by the range of temperature and pressures near thesupercritical point for a given supercritical fluid. Accordingly, one ofordinary skill in the art will be able to select the appropriate time,temperature, and pressure ranged based upon the supercritical fluidused, the size or amount of outer particle expansion desired, and thedegree of porosity in the outer particle desired. For example, exposurefor longer periods of time and/or at higher pressures followed bypressure quench will result in greater expansion and porosity thanshorter exposure times and/or pressures.

In one exemplary embodiment, the inner particles and outer particles aremixed at a ratio of approximately 1:3. In one exemplary embodiment, theratio of inner particles to outer particles used is approximately 1:9.These ratios will influence the extent of nanoparticle incorporation andslow release of the drug. In general, the larger the amount of innerparticles relative to outer particles the higher the amount of innerparticles incorporated in outer particle, increasing the drug releaserates and the dose. The smaller the amount of inner particles relativeto the outer particles, the smaller the burst release.

Protein Aggregation-Mediated Diseases and Methods of Treatment

The LEDGF formulations of the present invention may be used to treatprotein-aggregation mediated diseases. Protein aggregation-mediateddiseases that may be treated with the LEDGF compositions of the presentinvention include retinal degenerative diseases and neurodegenerativediseases. Retinal degenerative diseases include age related maculardegeneration retinitis pigmentosa, and diabetic retinopathy.Neurodegenerative diseases include Alzheimer's disease (AD), Parkinson'sdisease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis,and prion diseases.

In one exemplary embodiment, the present invention comprises methods oftreating a retinal degenerative disease comprising administering to apatient with a retinal degenerative disease a composition comprising aLEDGF peptide of the present invention. In certain exemplary embodimentsthe LEDGF peptide is SEQ OD NO: 2. In one exemplary embodiment, theLEDGF composition is delivered transclerrally. In another exemplaryembodiment, the LEDGF composition is administered to the eye topicallyin the form of eye drops. In another exemplary embodiment, the LEDGFcomposition is implanted or systemically administered in an extendedrelease formulation. In one exemplary embodiment, the extended releaseformulation is a nanoassembly extended release formulation, such as aLEDGF/zinc nanoassembly formulation. In another exemplary embodiment,the extended release formulation is a particle-in-particle formulation,such as a nanoparticle in porous microparticle (NPinPMP) formulation.

In one exemplary embodiment, the present invention comprises methods ofreducing protein aggregation in a retinal degenerative diseasecomprising administering to a patient with a retinal degenerativedisease a composition comprising a LEDGF peptide of the presentinvention. In certain exemplary embodiments the LEDGF peptide isLEDGF₁₋₃₂₆. In one exemplary embodiment, the LEDGF composition isdelivered transclerrally. In another exemplary embodiment, the LEDGFcomposition is administered to the eye topically in the form of eyedrops. In another exemplary embodiment, the LEDGF composition areimplanted or systemically administered in an extended releaseformulation. In one exemplary embodiment, the extended releaseformulation is a nanoparticle extended release formulation of thepresent invention, such as a LEDGF/zinc nanoparticle formulation. Inanother exemplary embodiment, the extended release formulation is aparticle-in-particles formulation, such as a nanoparticle in porousmicroparticle (NPinPMP) formulation.

In another exemplary embodiment, the present invention comprises methodsof treating neurodegenerative diseases comprising administering to apatient with a neurodegenerative disease a composition comprising aLEDGF peptide of the present invention. In certain exemplaryembodiments, the LEDGF peptide is LEDGF₁₋₃₂₆. In one exemplaryembodiment, the LEDGF composition is delivered intraperitoneally. Inanother exemplary embodiment, the LEDGF composition is administeredorally. In another exemplary embodiment, the LEDGF composition areadministered systemically in an extended release formulation. In oneexemplary embodiment, the extended release formulation is a nanoparticleextended release formulation of the present invention, such as aLEDGF/zinc nanoparticle formulation. In another exemplary embodiment,the extended release formulation is a particle-in-particle formulation,such as a nanoparticle in porous microparticle (NPinPMP) formulation.

In another exemplary embodiment, the present invention comprises methodsof reducing protein aggregation in a neurodegenerative diseasecomprising administering to a patient with a neurodegenerative disease acomposition comprising a LEDGF peptide of the present invention. Incertain exemplary embodiments, the LEDGF peptide is LEDGF₁₋₃₂₆. In oneexemplary embodiment, the LEDGF composition is deliveredintraperitoneally. In another exemplary embodiment, the LEDGFcomposition is administered orally. In another exemplary embodiment, theLEDGF composition is administered in an extended release formulation. Inone exemplary embodiment, the extended release formulation is ananoparticle extended release formulation of the present invention, suchas a LEDGF/zinc nanoparticle formulation. In another exemplaryembodiment, the extended release formulation is a particle-in-particleformulation, such as a nanoparticle in porous microparticle (NPinPMP)formulation.

The compositions and methods are further illustrated by the followingnon-limiting examples, which are not to be construed in any way asimposing limitations upon the scope thereof. On the contrary, it is tobe clearly understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which, after readingthe description herein, may suggest themselves to those skilled in theart without departing from the spirit of the present invention.

EXAMPLES Example 1 1. Materials

Plasmid pEGFP-LEDGF was gifted by Dr. Toshimichi Shinohara (Universityof Nebraska Medical Center, Omaha, Nebr.). Forward and reverse primerswere obtained from Integrated DNA Technologies (Coralville, Iowa). DNApolymerase I, T4 DNA ligase, and restriction enzymes were obtained fromNew England Biolab Inc. (Ipswich, Mass.). QIAquick gel extraction kit,QIAprep spin miniprep kit, and QIAGEN plasmid mini kit were obtainedfrom Qiagen (Valencia, Calif.). XK 16/20 column, S-200 gel filtrationcolumn, SP sepharose beads were obtained from GE Lifesciences Healthcare(Piscataway, N.J.). ARPE-19 cells were obtained from ATCC (Manassas,Va.). DMEM/F12 cell culture medium, fetal bovine serum, Lipofectamine2000, LB medium, and ultra-pure agarose were obtained from Invitrogen(Carlsbad, Calif.). All other materials unless specified were obtainedfrom Sigma-Aldrich (St. Louis, Mo.).

Preparations of LEDGF₁₋₃₂₆ DNA Construct

Gene encoding LEDGF₁₋₃₂₆ protein was cloned into a pET-28 a (+) vector(Novagen, Madison, Wis.). Briefly, the Ledgf₁₋₃₂₆ gene was PCR(polymerase chain reaction) amplified from the pEGFP-LEDGF plasmid usingthe forward primer 5′AGTAGTGGATCCATGACTCGCGATTTCAAAC3′ (SEQ ID NO: 3)consisting of HindIII restriction endonuclease site and reverse primer5′AATAATAAGCTTTCACTGCTCAGTTTCCATTTGTTC′3 (SEQ ID NO: 4) consisting ofBamHI restriction endonuclease site. PCR amplification was done usingDNA polymerase I at 94° C. denaturation for 5 min followed by 36 cyclesof denaturation at 94° C. for 30 secs, annealing at 50° C. for 45 secs,and extension at 72° C. for 2 min, and a final step of extension at 72°C. for 5 min. The amplified Ledgf₁₋₃₂₆ gene was purified using theQIAquick gel extraction kit as per manufacturer's protocol. Thereafterthe purified Ledgf₁₋₃₂₆ gene insert and pET-28a(+) vector were seriallydigested at 5′ and 3′ end using HindIII and BamHI restriction enzymesrespectively. They were then purified using a QIAprep spin miniprep kitas per manufacturer's protocol. The sticky ends of the insert and thevector were ligated overnight at 4° C. using T4 DNA ligase. CompetentEscherichia coli DH5α cells were transformed with the ligation productusing heat shock procedure as per the manufacturer's protocol. Tencolonies were picked and the plasmid was amplified, extracted, andpurified using the QIAGEN plasmid mini kit. Insertion of the Ledgf₁₋₃₂₆in pET-28a(+) vector was confirmed by three different ways, first by PCRscreening, second by restriction digest, and finally by DNA sequencing.Purity and the size of the recombinant DNA was analyzed using 2% agarosegel. DNA quantifications were done using NanoDrop 1000 (Thermoscientific, Wilmington, Del.). The colony showing positive PCR signaland correct sequencing was cultured further and the bacterial glycerolstock was made and stored at −80° C. for all future use.

Bioinformatic Analysis

LEDGF₁₋₃₂₆ amino acid sequence was submitted to ExPASy bioinformaticsresource portal and the molecular weight, theoretical pI, amino acidcomposition, atomic composition, extinction coefficient, estimatedhalf-life of LEDGF₁₋₃₂₆ was computed.

Expression and Purification of LEDGF₁₋₃₂₆

For protein biosynthesis, pLEDGF₁₋₃₂₆ plasmid was amplified and purifiedfrom Escherichia coli DH5α colony using QIAGEN plasmid mini kit as permanufacturer's protocol. The plasmid was then transformed in Escherichiacoli BL21(DE3) strains as per manufacturer's protocol. Thereafter, asingle colony of the bacteria containing the plasmid was inoculated intoLB (Luria broth) medium containing 50 μg/ml of kanamycin overnight. A 1%inoculum of overnight grown culture was added to one liter of LB mediumcontaining 50 μg/ml of kanamycin. The culture was allowed to grow at 37°C. until the optical density (O.D.) of 0.6-0.8 was reached for theculture medium. Protein expression was induced by adding IPTG(Isopropyl-β-D-thio-galactoside) to the final concentration of 200 μM.Thereafter, cells were further incubated for 3 hours at 37° C. andharvested by centrifugation at 3000 g for 15 min at 4° C. Harvestedcells were resuspended in buffer A (25 mM Tris-HCl pH 7.0, 1 mM EDTA, 1mM PMSF, and 5% sucrose). Cells were pulse sonicated (Mesonix, Sonicator3000, Farmingdale, N.Y.) at 70% output (36 watt) for 5 secs followed bycooling for 15 secs for total of 30 min. Lysed cells were centrifuged at13000 g for 20 min at 4° C. to separate the soluble and insolublefractions of the lysate. The soluble (supernatant) and insoluble(pellet) fractions were analyzed on SDS-PAGE for protein content anddetermination of soluble/insoluble nature of produced protein.

FPLC:

LEDGF₁₋₃₂₆ was solely expressed in soluble fraction as determined bySDS-PAGE. LEDGF₁₋₃₂₆ was purified using fast protein liquidchromatography (FPLC) technique in two steps, first based on charge(cation exchange) and then based on size (gel filtration). Briefly,cation exchange SP sepharose beads were packed in XK 16/20 column andequilibrated using buffer A at 2 ml/min flow rate. Thereafter, thesoluble fraction was loaded on the column at flow rate of 1 ml/min. Thecolumn was then washed with five column volume of buffer A at 2 ml/minflow rate. Most of the non-specifically and loosely bound impuritieswere eluted using a sharp gradient of sodium chloride (0 to 28%conductance). After removal of significant proportion of column boundimpurities, further elution of LEDGF₁₋₃₂₆ was achieved using secondgradient of NaCl from 28 to 40% conductance in 40 min. The proteinelution profile was monitored by measuring absorbance at 280 nm using aninbuilt UV detector. Fractions of 2.5 ml were collected during elutionprocess and analyzed on SDS-PAGE to determine purity. Fractionscontaining high protein amount were pooled together. The pooledfractions were dialyzed using dialysis buffer (25 mM Tris pH 7.0, and0.1% sucrose) and then lyophilized for 48 hours. The lyophilized proteinwas solubilized in 2 ml of D.I. water. For the next step ofpurification, pre-packed S-200 gel filtration column was equilibratedusing the equilibration buffer B (25 mM Tris-HCl pH 7.0, and 100 mMNaCl) at flow rate of 1 ml/min. The LEDGF₁₋₃₂₆ concentrated solutionfrom the cation exchange was then loaded onto the S-200 column.LEDGF₁₋₃₂₆ was eluted using the buffer B at a flow rate of 1 ml/min. Asharp peak was obtained at about 100 min, fractions of 1 ml werecollected. The collected fractions were analyzed using SDS-PAGE.Fractions containing the pure LEDGF₁₋₃₂₆ were pooled together. Thepurified LEDGF₁₋₃₂₆ was then dialyzed extensively in the dialysis buffer(25 mM Tris-HCl pH 7.0, and 0.1% Sucrose), for 48 hours at 4° C. withthree buffer exchanges, to remove excess salt and other impurities. Thedialyzed LEDGF₁₋₃₂₆ was frozen and lyophilized for 48 hours at −80° C.The lyophilized LEDGF₁₋₃₂₆ was aliquoted and stored at −80° C. for allfuture purposes.

UV spectroscopy:

Twelve mg of the lyophilized LEDGF₁₋₃₂₆ was accurately weighed and thendissolved in 1 ml of D.I. water. UV absorbance spectrum was recordedfrom 200 nm to 400 nm using Spectramax M5 (Molecular Devices,Downingtown, Pa.). The sample was serially diluted using D.I. wateruntil absorbance of less than 1 was obtained at 280 nm. This absorbancewas used to calculate the amount of purified protein present in thesample based on the molar extinction coefficient.

Physical Characterization

The molecular weight and the purity of the LEDGF₁₋₃₂₆ protein weredetermined by SDS-PAGE, SEC-HPLC, and MALDI-TOF.

SDS-PAGE:

Briefly 5 μg of LEDGF₁₋₃₂₆ was boiled for 10 min in SDS-PAGE samplebuffer (containing beta-mercaptoethanol). The protein sample was loadedonto 4-15% SDS-PAGE gel (Bio-Rad, Hercules, Calif.) and electrophoresedfor 90 min at 30 mA. The gel was then stained with coomassie brilliantblue and visualized under white light using GelDoc™ XR (Biorad,Hercules, Calif.). For non-reducing SDS-PAGE, LEDGF₁₋₃₂₆ was diluted innon-reducing sample buffer (no beta-mercaptoethanol) and boiling wasavoided.

SEC-HPLC:

The lyophilized protein was dissolved in D.I. water to finalconcentration of 500 μg/ml and filtered through 0.22 um (PVDF) filter.The protein was assessed using Agilent Bio SEC-3 column using 25 mM Trisbuffer containing 1 mM CaCl₂, pH 7.0 at 25° C. with a flow rate of 1ml/min. The column was calibrated with molecular weight standards(Invitrogen). UV-absorbance was measured at 214 nm.

MALDI-TOF:

Protein homogeneity and identity was confirmed by 4800 Plus MALDITOF/TOF™ (AB Sciex, Framingham, Mass.) by determining the molecularweight. The protein sample was dissolved into a solution of standardMALDI matrix sinnapinic acid, spotted and dried onto the metal targetplate. Data were collected as total ion current (TIC) from 1000 lasershots of 5900 intensity.

DLS:

The homogeneity and size of the LEDGF₁₋₃₂₆ protein was analyzed usingzetasizer Nano ZS (Malvern, Westborough, Mass.). Briefly, lyophilizedprotein sample was dissolved in D.I. water to get final proteinconcentration of 2.5 mg/ml. Size was measured in terms of number,intensity and volume means using the dynamic light scattering techniquewith data collection at 173° backscatter angle. Measurement was anaverage of 13 scans.

Biophysical Characterization

For biophysical characterization the protein was extensively dialyzed in25 mM phosphate buffer pH 7.0 to remove Tris-HCl and sucrose andfiltered through 0.22 μm PVDF syringe filter. Spectra obtained wereanalyzed using either Origin® 8.5 (OriginLab Corp., Northampton, Mass.)or SigmaPlot 11.0 (Systat Software, Inc, Chicago, Ill.). The data wasfitted using equations 1 and 2, defined by Scholtz et. al. as below todetermine the ΔG, m-value, and [urea]_(1/2) [62].

$y = \frac{\begin{Bmatrix}{\left( {\overset{\Diamond}{y_{F}} + {m_{F}\lbrack{urea}\rbrack}} \right) +} \\{\left( {\overset{\Diamond}{y_{U}} + {m_{F}\lbrack{urea}\rbrack}} \right) \times} \\^{- {(\frac{{\Delta \; {G{({H_{2}O})}}} - {m{\lbrack{urea}\rbrack}}}{RT})}}\end{Bmatrix}}{1 + ^{- {(\frac{{\Delta \; {G{({H_{2}O})}}} - {m{\lbrack{urea}\rbrack}}}{RT})}}}$Δ G = Δ G(H₂O) − m[urea]

where yF° and yU° are the intercepts, m where y_(F) ^(⋄)and y_(U)^(⋄)are the intercepts, m_(F) and m_(U) are the slopes of the pre- andpost-transition phase baselines, and m-value is the slope of thetransition phase. ΔG is the free energy change at any particular ureaconcentration and it varies linearly with urea concentration, and isused to estimate ΔG(H₂O). ΔG(H₂O) is defined as the conformationalstability of a protein in the absence of urea at 25° C. R is universalgas constant and T is the temperature of the sample. [urea]_(1/2) is theconcentration of urea at which LEDGF₁₋₃₂₆ is 50% folded and 50%unfolded.

CD:

To determine the secondary structures of LEDGF₁₋₃₂₆ and determine itsconformational stability parameters, far-UV CD spectrum of the dialyzedprotein was recorded. Briefly, 500 μg/ml of protein sample was placed in1 mm quartz cuvette and spectra was recorded at a scan speed of 0.5 secsper time point, step size of 1 nm and the bandwidth of 4 nm from 200 to280 nm using Chirascan® CD instrument (Applied Photophysics Ltd, UK).All scans were done in triplicate. The native LEDGF₁₋₃₂₆ spectrum thusobtained was deconvulated using CDNN 2.1 CD spectra deconvulationsoftware (Dr. Gerald Bohm, Martin-Luther-University at Halle,Wittenberg, Germany, UK) to get the percentage of various secondarystructures present in native LEDGF₁₋₃₂₆ protein. For conformationalstability of LEDGF₁₋₃₂₆, chemical denaturation was performed at variousurea concentrations. Briefly, 300 μg/ml of protein was incubated with 0to 6 M urea in 25 mM phosphate buffer pH 7.0 for 24 hours. CD signal wasrecorded as mentioned above. The conformational stability parameters ofLEDGF₁₋₃₂₆ were determined by plotting the CD signal at 230 nm as afunction of urea concentration to obtain the maximum CD signaldifference between the folded and unfolded protein spectrum at thiswavelength. Similarly, to investigate the thermal stability ofLEDGF₁₋₃₂₆, 500 μg/ml of LEDGF₁₋₃₂₆ was subjected to heat denaturationfrom 25° C. to 90° C. in smooth ramp mode at ramp rate of 1° C. per min.The CD signal at 222 nm was used to determine the melting point (T_(m)).

Fluorescence Spectroscopy:

Steady state fluorescence spectroscopy was done to determine thetertiary structure perturbation. The protein sample (final concentration300 μg/ml) was incubated with various concentration of urea solution (0to 6 M) in 25 mM phosphate buffer pH 7.0 for 24 hours. The intrinsictryptophan fluorescence spectra were recorded from 300 to 400 nm, at 280nm excitation wavelength, with an increment of 1 nm using Spectramax M5(Molecular Devices, Downingtown, Pa.). The conformational stabilityparameters of LEDGF₁₋₃₂₆ were determined by plotting the fluorescenceintensity ratio at 340/356 nm as a function of urea concentration. Allintensity values were corrected for buffer effects and inner filtereffects.

Cell Viability Assay

ARPE-19 cells were used to determine the cell survival function ofLEDGF₁₋₃₂₆ in presence of aggregation mediated stress. Briefly, ARPE-19cells were maintained as describer earlier (Baid et al., PLoS One. 6(9):p. e24616). For cell viability assay, 10,000 cells were plated in96-well plate and incubated for 24 hours. After 24 hours, the serumcontaining medium was aspirated out. The test groups (pP23H-Rho+LEDGF₁₋₃₂₆) were transiently transfected with pP23H-Rho plasmid (1μg/ml) using 1:3 ratio of lipofectamine 2000 in serum free medium as permanufacturer's protocol. After six hours of transfection, the mediumwere aspirated out and cells were treated with increasing amount ofLEDGF₁₋₃₂₆ with the concentration range of 0.001 μg/ml to 50 μg/ml for48 hours. No cells (just the medium), cells with no lipofectamine 2000and cells with lipofectamine 2000 were also maintained as control. After48 hours, the medium was aspirated out and 200 μl of fresh serum freemedium was added. 20 μl of MTT reagent(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide, 5 mg/mlin PBS pH 7.4) was added to each well and further incubation was donefor 3 hours at 37° C. The MTT containing medium was aspirated out andthe formazan crystal formed was dissolved in 200 μl of DMSO. Theabsorbance of the color developed was measured at 570 nm usingSpectramax M5. The percentage viability of groups was calculated withreference to the control group containing cells with no lipofectamine2000. All groups were repeated as n=4.

Animal Maintenance

A RCS rat colony was maintained in the animal facility of University ofColorado Anschutz Medical Campus and with approval of IUCAC. Theexperiments were carried as per the ARVO statement for the Use of theAnimals in Ophthalmic and Vision Research.

Electroretinography

At 4 weeks age, rats were dark adapted for 30 min. Thereafter, theanimal was prepared for ERG under dim red light. Briefly, the rat wasanaesthetized with intraperitoneal injection of mixture of 80 mg/kg ofketamine and 12 mg/kg of xylazine. The pupil was then dilated with adrop of 0.5% tropicanamide (Akorn, Lake Forest, Ill.) and was kept moistusing a drop of 2.5% hypromellose (Akorn, Lake Forest, Ill.).Thereafter, the animal was placed on a heated water jacket stabilized at37° C. A reference electrode (LKC Technologies Inc., Gaithersburg, Md.)was inserted into the animal's tail and cheek. A DTL plus electrode (LKCTechnologies Inc., Gaithersburg, Md.) was placed across the cornea ofeach eye. Each animal was exposed to brief flashes of 0.4 log cd-s/m2with interval of 10 secs between each flash and scotopic ERG wasrecorded. Thereafter, the animal was light adapted for 3 min with abackground light of 30 cd/m². Photopic ERG was recorded at sameintensity flash but with background light on. At least three ERGs wereaveraged to get a single ERG for each rat. Thereafter, sterile filtered,2 μl of 0.25, 0.5, or 2.5 mg/ml of LEDGF₁₋₃₂₆ was given intravitreallyin one eye and vehicle in contralateral eye. ERGs were recorded everytwo weeks for 8 weeks after intravitreal injection, i.e. till week 12age of rats.

Statistical Analysis

Data are represented as the mean±SD. An independent-samples student'st-test or one-way ANOVA followed by Tukey's post hoc analysis (SPSS,ver.11.5; SPSS, Chicago, Ill.) was performed for comparisons between thetwo or multiple experimental groups, respectively. The differences wereconsidered statistically significant at p≦0.05.

2. Results

LEDGF₁₋₃₂₆ Cloned into pET28a(+)

DNA fragment of about 1000 base pairs was obtained from the polymerasechain reaction (PCR) amplification of Ledgf₁₋₃₂₆ gene (FIG. 3, Lane 1)from pEGFP-LEDGF plasmid. The undigested pET-28 a (+) vector (Lane 2)showed a positive band at about 4.5 kb, which when digested using BamHIgot linearized and shifted upward in between 5 and 6 kb DNA marker (Lane3). When Ledgf₁₋₃₂₆ gene was ligated in pET-28a (+) vector (the plasmidso obtained will be designated as pLEDGF₁₋₃₂₆ from here onwards), therewas an upward band shift equivalent to ˜1000 bp, indicating successfulinsertion of Ledgf₁₋₃₂₆ gene in the pET28a(+) vector (Lane 4). Toconfirm the ligation, pLEDGF₁₋₃₂₆ was singly digested either with BamHI(Lane 5) or HindIII (Lane 6). pLEDGF₁₋₃₂₆ was linearized by bothrestriction enzyme digestion reactions and the DNA band was seen atabout 6.4 kb which was 1000 bp more than pET28a(+) vector. Double digestof pLEDGF₁₋₃₂₆ using BamHI and HindIII resulted in two fragments, abigger fragment of ˜5.4 kb (upper band, Lane 8) and a smaller fragmentof ˜1000 bp (lower band, Lane 7). PCR amplification of Ledgf₁₋₃₂₆ genefrom pLEDGF₁₋₃₂₆ resulted in a positive DNA band of about 1000 bases(Lane 8), indicating that LEDGF₁₋₃₂₆ was inserted successfully in pET-28a (+) vector.

Bioinformatic Analysis of LEDGF₁₋₃₂₆

Bioinformatics analysis of LEDGF₁₋₃₂₆ sequence using SIB ExPASy(Gasteiger et al., Nucleic Acids Res, 2003. 31(13): p. 3784-8) indicatedits theoretical molecular weight of 36.9 kDa. The computed isoelectricpoint (pI) of LEDGF₁₋₃₂₆ was 9.23, with 73 positively charged (arginineand lysine) and 63 negatively charged (aspartic acid and glutamic acid)amino acid residues. The theoretical molar extinction coefficient was15470 M⁻¹ cm⁻¹ at 280 nm in water. Based on its N-terminal amino acidmethionine, its half-life in mammalian cells was predicted to be 30hours.

LEDGF₁₋₃₂₆ Purified in Bulk Quantities

A new strong positive band appeared, at about 40 kDa, when LEDGF₁₋₃₂₆was expressed under specified conditions in BL21(D3B) cells indicatingexpression of LEDGF₁₋₃₂₆ protein (FIG. 4A, Lane 3). This band appearedin the supernatant fraction after the bacterial cell lysis, indicatingthat LEDGF₁₋₃₂₆ was expressed as soluble protein in bacterial culture(Lane 4). LEDGF₁₋₃₂₆ was purified from the crude cell lysate using fastprotein liquid chromatography (FPLC) system. In first step ofpurification, cation exchange column was used (FIG. 4B). The unboundprotein and other cellular impurities including lipids got eluted duringthe column washing phase (100-280 ml). Thereafter, other cellularproteins which were loosely bound to the column got eluted out when theconductance of the mobile phase was increased from 0 to 28% using sharpsodium chloride (NaCl) gradient (280-400 ml). When the gradient of NaClwas further increased slowly over 40 min to reach 40% conductance,LEDGF₁₋₃₂₆ was eluted (400-450 ml). When the collected fraction wasanalyzed using SDS-PAGE, a strong band of LEDGF₁₋₃₂₆ at ˜40 kDa was seenalong with other lower molecular weight bands (FIG. 4A, Lane 5). Onfurther purification using the gel filtration column, LEDGF₁₋₃₂₆ waseluted as the first peak (fractions collected), followed by peaks ofother proteins of smaller molecular weights (FIG. 4C). Pooled fractionsfrom the gel filtration columns indicated a strong positive band of ˜40kDa in SDS-PAGE along with very faint low molecular weight bandsindicating almost complete purification of LEDGF₁₋₃₂₆ (FIG. 4A, Lane 6).Protein estimation indicated that about 20 mg of protein was obtainedper liter of the shake flask culture.

LEDGF₁₋₃₂₆ is Purified to Near Homogeneity

The purity of LEDGF₁₋₃₂₆ protein was determined by size exclusionchromatography (SEC-HPLC) (FIG. 5A). Two peaks were observed, first peakhad a retention time of 10.5 min and the second peak had the retentiontime of about 11.5 min. When the area under the curve was integrated,the first peak was only 5% and the second peak was 95%, indicating thatLEDGF₁₋₃₂₆ protein was about 95% pure. The molecular weight ofLEDGF₁₋₃₂₆ was confirmed by matrix assisted laser desorption/ionization(MALDI-TOF). The major peak obtained in MALDI-TOF spectrum was at40314.32 and 80663.19 m/z (mass to charge) ratio (FIG. 5B). MALDI-TOFindicated that LEDGF₁₋₃₂₆ has a molecular weight of 40 kDa, which wasequivalent to theoretical molecular weight. A second peak at 80663 m/zwas also seen, which indicated that LEDGF₁₋₃₂₆ may exist as dimer. Toinvestigate the existence of the dimers, SDS-PAGE of LEDGF₁₋₃₂₆ was rununder reducing and non-reducing conditions (FIG. 5D). Under non-reducingconditions, there was an upward shift of the LEDGF₁₋₃₂₆ band to 95-105kDa size, indicating that LEDGF₁₋₃₂₆ may exist in dimeric form. Underreducing/denaturing conditions the dimers dissociated into monomers.

To further investigate whether LEDGF₁₋₃₂₆ self-associates to form anyhigher molecular weight oligomers, dynamic light scattering (DLS) wasperformed (FIG. 5C). A homologous population having a single peak (100%distribution) was obtained for intensity or number or volume sizedistribution of the particles. All three peaks were in the narrow sizedistribution indicating a very close size range. There were no otherpeaks of higher sizes indicating absence of any kind of oligomers. Thez-average diameter of 11.32 nm, and a polydispersity index of 0.15 wasobtained from DLS.

LEDGF₁₋₃₂₆ is Randomly Coiled

To investigate the secondary structure of LEDGF₁₋₃₂₆, far-UV circulardichroism (CD) spectrum of the native LEDGF₁₋₃₂₆ was analyzed (FIG. 6A).The CD signal remained negative from 280 to 200 nm. After 220 nm, therewas a steep decline in the CD signal. There were no characteristicnegative bands at 222 nm and 208 nm, neither there was anycharacteristic positive band near 200 nm indicating that LEDGF₁₋₃₂₆ donot possess predominantly α-helix or β-sheets. In fact very lowellipticity above 210 nm and negative band below 200 nm indicated thatthe LEDGF₁₋₃₂₆ may be composed of primarily random coils.

To further dissect the secondary structure of LEDGF₁₋₃₂₆, the CDspectrum was deconvulated using CDNN 2.1 software. Assuming that thespectrum obtained is the linear combination of the individual spectrumof the component secondary structure elements and noise due to thearomatic chromophores and prosthetic groups, LEDGF₁₋₃₂₆ was predicted tobe 45.1% randomly coiled. The β-turn was about 21.2%, there were 15%parallel β-sheets and 16% antiparallel β-sheets. The contribution fromthe α-helix was about only 16%.

The three dimensional structure of LEDGF₁₋₃₂₆ native protein waspredicted using I-Tasser (Iterative Threading Assembly Refinement)protein modeling server (FIG. 6B). LEDGF₁₋₃₂₆ predicted model had theconfidence score (C-score) of −3.18, Template modeling (TM-score) of0.36±0.12, and root mean square deviation (RMSD) was equal to 14.1±8 Å,indicating that the predicted model is reliable. According to thepredicted model, LEDGF₁₋₃₂₆ was predominantly a random coiled protein.

LEDGF₁₋₃₂₆ is Conformationally Stable

To understand the conformational stability of LEDGF₁₋₃₂₆ in water, theperturbation in the tertiary structure due to chemical denaturation wasdetermined by measuring the intrinsic fluorescence of tryptophanmolecules present in LEDGF₁₋₃₂₆ (FIGS. 7A and 7B). Emission spectrum ofnative LEDGF₁₋₃₂₆ protein, in absence of urea, had a λmax at 340 nm andΔλ_(1/2) (half width of Δλ) of 56 nm (FIG. 7A). As the concentration ofurea increased from 0 to 5 M, quenching in the fluorescence signal aswell as red shift (fluorescence maxima shifting towards right) was seen.The signal decreased slowly until 0.9 M urea concentration was reached.Thereafter, there was a sharp decrease in the fluorescence signal until2.3 M urea concentration was reached. Beyond this concentration, thedecrease in the fluorescence signal was minimal. The λ_(max) ofLEDGF₁₋₃₂₆ shifted to 356 nm and Δλ_(1/2) was 71 nm at 5 M urea. Whenthe ratio of LEDGF₁₋₃₂₆ fluorescence signal at 340 to 356 nm was plottedas a function of urea concentration, a sigmoidal curve was obtained(FIG. 7B). There was a slow decline of fluorescence signal from 0-1 Murea (pre transition phase) and then a steep decay from 1-3 M urea(transition phase) followed by a slow decline phase from 3-5 M (posttransition phase). Using the equations 1 and 2 (described in methods),ΔG(H₂O) of LEDGF₁₋₃₂₆ was estimated to be 3.24±0.48 kcal mol⁻¹, them-value to be 1.70±0.22 kcal mol⁻¹M⁻¹, and [urea]_(1/2) to be 1.81±0.02M, indicating that LEDGF₁₋₃₂₆ is a stable protein.

Far-UV CD spectroscopy was performed to investigate the perturbation inthe secondary structures of LEDGF₁₋₃₂₆ in presence of urea (FIGS. 7C and7D). The CD signal of the LEDGF₁₋₃₂₆ was traced against the wavelengthat each urea concentration (FIG. 7C). The CD signal continuously becamemore negative as the concentration of urea was increased. When CD signalat 230 nm was plotted as a function of urea concentration (FIG. 7D), asigmoidal curve was obtained. Fitting the data in equations 1 and 2,indicated ΔG(H₂O) of LEDGF₁₋₃₂₆ to be 3.28±0.40 kcal mol⁻¹, m-value of1.90±0.19 kcal mol⁻¹M⁻¹, and [urea]_(1/2) to be of 1.61±0.02 M.

LEDGF₁₋₃₂₆ is Thermally Stable

Thermal stability of LEDGF₁₋₃₂₆ was determined using far-UV CDspectroscopy (FIGS. 7E and 7F). The CD signal in the presence of heat asa denaturant was measured from 215-250 nm (FIG. 7E). As the temperatureof the LEDGF₁₋₃₂₆ solution was increased, the negative dip obtained atabout 235 nm was seen to increase. The CD signal followed the samepattern as chemical denaturation, a pre-transition phase between ˜30-35°C., followed by transition phase between ˜35-55° C., followed bypost-transition phase from ˜55-70° C. (FIG. 7F). When this data wasfitted using a global fit analysis, the T_(m) (the melting temperature)of LEDGF₁₋₃₂₆ obtained was 44.82±0.17° C. indicating LEDGF₁₋₃₂₆ willpossibly be stable at 25° C. (room temperature).

LEDGF₁₋₃₂₆ Rescues ARPE-19 Cells from Aggregation Mediated Stress

LEDGF₁₋₃₂₆ activity to rescue ARPE-19 cells from protein aggregationmediated stress was measured by cell viability assay (FIG. 8).Initially, the ability of LEDGF₁₋₃₂₆ to increase the viability ofARPE-19 cells in absence of any stress was investigated (FIG. 8A). Therewas no significant difference in the cell viability in untreated and0.001 to 50 μg/ml LEDGF₁₋₃₂₆ treated cells following 48 hr treatment. Atthe highest dose of LEDGF₁₋₃₂₆ (50 μg/ml), the cell viability was108.14±5.63% (right most bar) as compared to 100±13.19% for untreatedcells (left most bar), which was not significant. In pP23H-Rhotransfected ARPE-19 cells, LEDGF₁₋₃₂₆ behaved differently (FIG. 8B).Cells expressing P23H mutant rhodopsin showed a decline in cellviability to 48.25±5.62% (Bar 2). This loss in cell viability could beattributed to toxic effect of expression and accumulation of aggregationprone P23H mutant rhodopsin protein within the cells. When cellsexpressing P23H mutant rhodopsin (Bar 3-9) were treated with increasingamount of LEDGF₁₋₃₂₆, an increase in the cell viability was seen.LEDGF₁₋₃₂₆ increased the cell viability of ARPE-19 cells at as lowconcentration as 0.001 μg/ml from 48.25±5.62 to 77.02±10. 20%. Beyondthis point the cell viability remained significantly higher than theuntreated pP23H-Rho transfected group (Bar 2).

LEDGF₁₋₃₂₆ Delays the Functional Loss of Photoreceptors

LEDGF₁₋₃₂₆ efficacy to delay the loss of visual function ofphotoreceptors was investigated in RCS rats by monitoring theelectroretinograms (ERG). In dark adapted (scotopic) ERG, the b-waveamplitude of untreated and treated rats ranged from 180.17±27.42 to216.60±35.30 μV at 4 weeks age (base ERG), before intravitreal injectionwas administered (FIG. 9A). At two weeks after intravitreal injection,there was a sharp decline in the b-wave amplitude in all groups, thevalue ranged from 65.80±15.44 to 91.13±13.94 μV. There was nosignificant difference in the untreated and LEDGF₁₋₃₂₆ treated groups.However, beyond two weeks, there was a continuous decline in the b-waveamplitude in all groups, with decline being slower in the LEDGF₁₋₃₂₆treated groups. At eight weeks after the intravitreal injection, theb-wave amplitude of untreated, 0.5, 1.0, and 5 μg of LEDGF₁₋₃₂₆ treatedgroups was 9.40±4.57, 32.43±10.34, 37.93±0.60, and 57.63±8.81 μV,respectively. B-wave amplitude of LEDGF₁₋₃₂₆ treated groups weresignificantly (p<0.05) higher than the untreated group. A dose dependentdelay in the b-wave amplitude decline was seen for the LEDGF₁₋₃₂₆treated groups. With increasing dose of LEDGF₁₋₃₂₆, the loss of b-waveamplitude was reduced.

In light adapted (photopic) ERG, the base b-wave amplitude, beforeintravitreal injection, at week 4, was in range of 69.83±16.49 to80.97±8.60 μV, with no significant difference between the untreated andLEDGF₁₋₃₂₆ treated groups (FIG. 9B). The b-wave amplitude of untreatedgroup declined from 80.97±8.60 to 10.90±5.64 μV, whereas the decline wasfrom 79.63±20.30 to 41.33±9.20, 69.83±16.49 to 28.00±7.23, and68.75±15.93 to 45.78±15.18 μV for 0.5, 1.0, and 5.0 μg of LEDGF₁₋₃₂₆treated groups, respectively. B-wave amplitude of LEDGF₁₋₃₂₆ treatedgroups were significantly (p<0.05) higher than the untreated group aftereight weeks of single intravitreal injection of LEDGF₁₋₃₂₆ similar toscotopic ERG.

Example 2 1. Materials and Methods

Materials—

Isopropyl-β-D-thio-galactoside (IPTG) citric acid, dibasic sodiumhydrogen phosphate, ethylene diamine tetra acetic acid (EDTA), Tween 20,sucrose, sodium azide were purchased from Sigma-Aldrich( ). AKTA FLPCwas used for protein purification. All chromatograms were analyzed usingUNICORN software. All chemicals unless specified was obtained from SigmaAldrich and were of reagent or higher grade.

His-Tag Removal—

LEDGF₁₋₃₂₆ gene was amplified from pLEDGF₁₋₃₂₆ plasmid using5′AGCAAGCCATGGGCATGACTCGCGATTTCAAACCTGGA3′ (SEQ ID NO: 5) and 5′AGCAAGAAGCTTCTACTGCTCAGTTTCCATTTGTTCCTC3′ (SEQ ID NO: 6) primerscontaining NcoI and HindIII sites, respectively. The LEDGF₁₋₃₂₆ gene wasthereafter ligated into pET-28a (+) after digesting with Nco1 andHindIII enzymes. The ligated product was transformed in competentEscherichia coli DH5α cells as per user's manual. The insertion of thegene was confirmed by PCR, restriction digestion and sequencing methods.

LEDGF₁₋₃₂₆ Biosynthesis and Purification:

LEDGF₁₋₃₂₆ (His-tag free) was biosynthesized and purified as previouslydescribed (Ref-JBC). Briefly, LEDGF₁₋₃₂₆ was expressed in Escherichiacoli BL21 (DE3) using 1 mM IPTG. LEDGF₁₋₃₂₆ was purified from celllysates using two step fast protein liquid chromatography (FPLC), firstbased on charge (cation exchange) and then based on size (gelfiltration). The purified LEDGF₁₋₃₂₆ was extensively dialyzed incitrate-phosphate buffer pH 7.0, concentrated and stored at −80° C.until further use.

Formulation Preparation:

LEDGF₁₋₃₂₆ (1 or 0.5 mg/ml) formulation in citrate-phosphate buffer wasmade with pH ranging from 6 to 7.5. Additives Tween 20, EDTA, andsucrose was added to the final concentration of 0.1% (w/v), 1 mM, 10%(w/v), respectively. Formulation containing 0.02% sodium azide wastested for any degradation that might happen due to microbial growth.All formulations once prepared were stored at 25° C. in temperaturecontrolled incubator and adequate measure was taken to avoid anyevaporation.

Fluorescence Spectroscopy:

The steady state fluorescence spectroscopy was done to determine thechanges in the tertiary structure. The intrinsic tryptophan (Trp)fluorescence spectra of formulations were recorded from 300 to 400 nm,at 280 nm excitation, with every 1 nm increment using Spectramax M5(Molecular Devices, Downingtown, Pa.). To measure the changes in thefluorescence, fluorescence intensity at 342 nm was plotted for each pHand each time point. Buffer and inner filter effects were corrected forall fluorescence values.

Circular Dichroism (CD):

Secondary structural changes of LEDGF₁₋₃₂₆ was determined by far-UV CDspectra. Briefly, the formulation was placed in 1 mm quartz cuvette andspectra was recorded at a scan speed of 0.5 sec per time point, stepsize of 1 nm and the bandwidth of 4 nm from 200 to 280 nm usingChirascan® CD instrument (Applied Photophysics Ltd, UK).

Dynamic Light Scattering (DLS):

The size of the LEDGF₁₋₃₂₆ protein was monitored using Nano ZS (Malvern,Westborough, Mass.). Briefly 100 μl of formulation was placed in lowvolume glass cuvette. Using dynamic light scattering, LEDGF₁₋₃₂₆particle scattering data were collected at 173° backscatter angle. Anaverage of 13 scans were recorded for each measurement.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE):

The LEDGF₁₋₃₂₆ formulation samples (10 μg) were boiled for 10 min at 75°C. along with 10 μL of 2× loading buffer. Samples were loaded on 4-15%mini-PROTEAN TGX gels and proteins were size separated. Proteins werevisualized using Coomassie Blue staining as per user's protocol.

Protein Estimation:

For protein estimation, LEDGF₁₋₃₂₆ formulation was spin down at 10000 gfor 5 min and the supernatant was collected. Protein estimation ofsupernatant was done using BCA assay kit as per user's manual. Forinsoluble aggregate estimation, the soluble protein measured at eachtime point was subtracted from the day 0 protein levels of thecorresponding formulation.

ELISA:

An indirect ELISA method was developed to determine the percentage ofimmuno reactive LEDGF₁₋₃₂₆ in formulations. Briefly, in 96-well plate,100 μl of either standard LEDGF₁₋₃₂₆ (freshly purified) or formulationsamples were coated overnight at 4° C. in triplicates. Wells were washedthree times with wash buffer (0.1% w/v Tween 20 in PBS pH 7.0) aftereach step. The nonspecific binding sites were blocked with blockingsolution (0.5% bovine serum albumin, and 0.1% Tween 20 in PBS pH 7.0)for 4 hours. LEDGF₁₋₃₂₆ was detected by mouse anti-LEDGF antibody (BDBiosciences, San Diego, Calif.) which was cross detected with HRPconjugated anti-mouse secondary antibody (source). After through washingof the plate finally 3,3′,5,5′-Tetramethylbenzidine (TMB) was added.Immuno reactive LEDGF₁₋₃₂₆ was quantitate by colorimetric absorbance at650 nm upon development of blue color.

Statistical Analysis:

All data are represented as the mean±SD. For comparison between multiplegroups, data has been combined for all pH, averaged and compared tocorresponding additive containing formulations. Statistics was done byone-way ANOVA followed by Tukey's post hoc analysis (SPSS, ver.11.5;SPSS, Chicago, Ill.). p≦0.05 was considered to be statisticallysignificant.

2. Results His Tag Free LEDGF₁₋₃₂₆ Cloning and Purification

PCR amplification led to band of 1000 bp of LEDGF₁₋₃₂₆. Restrictiondigestion, ligation and subsequent PCR amplification from LEDGF₁₋₃₂₆gene inserted plasmid indicated a positive band of LEDGF₁₋₃₂₆.Purification of LEDGF₁₋₃₂₆ protein indicated a monomer band of 40 kDaalong with very faint small molecular weight bands indicating LEDGF₁₋₃₂₆might have undergone some degradation during purification process.

Additives Increases LEDGF₁₋₃₂₆ Stability

Effect of additives Tween 20, EDTA, and sucrose on LEDGF₁₋₃₂₆ stabilitywas monitored in citrate-phosphate buffer between pH 6.0-7.5. Thetertiary structure perturbation in LEDGF₁₋₃₂₆ was monitored by measuringthe fluorescence behavior of tryptophan (Trp) in LEDGF₁₋₃₂₆ (FIG. 11A).LEDGF₁₋₃₂₆ formulation (0.5 mg/ml) indicated no significant differencein fluorescence intensity at 342 nm with respect to pH of the buffer(FIG. 11 A(i)). On day 0 plain buffer formulations of LEDGF₁₋₃₂₆indicated initial fluorescence intensity of 5163±302 R.F.U. at 342 nm in6.0-7.5 pH range. On day 1 there was an increase in fluorescenceintensity to 6198±102 which decreased significantly to 3518±305 R.F.U.by day 3 indicating a loss of ˜43% signal intensity as compared today 1. By day 14 there was 87% loss in the signal intensity. Thefluorescence spectra indicated red shift in the maximum fluorescenceintensity on day 3, on day 7 and beyond the spectra were almost flat.

Additives did not change the initial LEDGF₁₋₃₂₆ fluorescence intensityand there was no significant difference in the fluorescence intensity offormulations with or without additives on day 0 (FIG. 11 A(ii)). Therewas no significant loss in the fluorescence signal as well as no shiftin the fluorescence maxima until day 60 for all pH range.

The secondary structure perturbation in LEDGF₁₋₃₂₆ was monitored by CD.FIG. 11B indicates the ellipticity of LEDGF₁₋₃₂₆ at 208 nm for variousformulations in 6.0-7.5 pH as function of time. CD indicated thesecondary structure of LEDGF₁₋₃₂₆ is primarily random coil. LEDGF₁₋₃₂₆indicated an ellipticity of −17.5±0.1 mDeg on day 0 which reducedsignificantly to −13.9±0.8 mDeg by day 3 (FIG. 11B(i)). There wasfurther decline in the CD signal and by day 7 onwards the ellipticitywas −3.4±2.3 mDeg. There was no significant difference in the CD signalin formulations with or without additives on day 0 (FIG. 11B(ii)). TheCD signal on day 0 and day 60 was −17.9±0.3 and −19.1±1.4 mDeg,respectively, for all formulations containing additives indicating therewere no significant changes.

The hydrodynamic (particle) size of LEDGF₁₋₃₂₆ was 7±1 nm on day 0 inall formulations with or without additives (FIG. 11C(i)) as indicated byDLS. By day 3 the particle size of LEDGF₁₋₃₂₆ increased to 200-700 nm inplain buffer formulations pH 6-7.5. In presence of additives, LEDGF₁₋₃₂₆indicated a size of ˜1 nm on day 0 and no change in size was indicateduntil day 60 (FIG. 11C(ii)).

SDS-PAGE indicated LEDGF₁₋₃₂₆ is a 40 kDa protein. On day 0 there werevery faint bands of small molecular weight proteins in all formulations.In plain buffer formulations the lower molecular weight fragmentsintensified as early as in day 1 (FIG. 12(i)). By day 3 there wassignificant amount of visible lower molecular weight bands. By day 7there was complete loss of 40 kDa band and other fragments. Additivesdelayed the intensification of lower molecular bands until day 60 (FIG.12(ii)). On day 60 lower molecular bands were visible along with the 40kDa band in additive containing formulation irrespective of buffer pH.

Additives Reduces Insoluble Aggregates of LEDGF₁₋₃₂₆

The soluble protein content on day 0 for LEDGF₁₋₃₂₆ plain bufferformulations pH 6.0-7.5 was 417.8±21.3 μg/ml (FIG. 13A). By day 7 therewas significant decrease in the soluble protein content to 142.5±60.7μg/ml. Thereafter, there was high variation in the protein content inplain buffer formulations at different pH, however there was no cleartrend. On an average by day 60 the total soluble protein content was316.2±140.0 μg/ml. Additives containing formulations indicated proteincontent of 470.5±17.3 μg/ml on day 0 and remained to be 469.0±33.4 μg/mleven until day 60.

Percentage aggregates present in the formulation was calculated from thesoluble protein content (FIG. 13B). Plain buffer formulations indicatedappearance of insoluble aggregates as early as day 3 and by day 7 therewas 64.6±14.0% aggregates (FIG. 13B). Beyond day 7 there wasunpredictable changes in the aggregate content in the formulation,however, presence of aggregates remained significantly high in all pHrange. In presence of additives, the percentage aggregates remainedbelow the detections limit for all days until day 60 except on day 30where there was 22.3±9% aggregates (FIG. 13B).

Particles that settled down in the formulations were visible in plainbuffer formulations (FIG. 13C), while the additive containingformulations were clear until day 60.

LEDGF₁₋₃₂₆ Remains Immunoreactive in Presence of Additives

LEDGF₁₋₃₂₆ immuno reactivity was quantified using an indirect ELISA(FIG. 14). ELISA indicated 76.9±4.8% immuno reactive LEDGF₁₋₃₂₆ on day 0in plain buffer formulations pH 6.0-7.5. On day 14 when immunoreactivitywas tested it was found that LEDGF₁₋₃₂₆ lost almost all of itsimmunoreactivity with only 3.1±2.4% remaining By day 60 immunoreactiveLEDGF₁₋₃₂₆ was undetectable. On the other hand additives containingformulations indicated 74.8±7.7, 66.2±2.8, and 70.4±24.5% immunoreactiveLEDGF₁₋₃₂₆ on day 0, day 14, and day 60, respectively. Theimmunoreactivity was seen to have pH dependency, while pH 6, and 7.5indicated 30±4 and 58±1% immunoreactive LEDGF₁₋₃₂₆, pH 6.5, 6.75, 7.0,and 7.25 indicated 78±15, 78±45, 76±9, and 102±13% immunoreactiveLEDGF₁₋₃₂₆ on day 60.

Individual Additives are Less Effective in Increasing LEDGF₁₋₃₂₆Stability

To understand the effect of individual additives on LEDGF₁₋₃₂₆ stabilityonly one additive at a time was tested in LEDGF₁₋₃₂₆ (1 mg/ml)formulations at pH 7.0 (FIGS. 15, and 16). Fluorescence intensity ofLEDGF₁₋₃₂₆ at 342 nm decreased from 8410±116 to 2178±22 R.F.U for plaincitrate-phosphate buffer formulation by day 30 (FIG. 15A). While for0.01% Tween 20, 1 mM EDTA, and 10% sucrose containing formulations, thefluorescence intensity was 4925±1249, 4056±979, and 6370±592 R.F.U.,respectively on day 30. Sodium azide used as control to monitor themicrobial contamination indicated fluorescence intensity of 9136±241R.F.U. In absence of additive, LEDG₁₋₃₂₆ fluorescence signal lost was75% of day 0 signal, Tween 20, EDTA, and sucrose retained thefluorescence signal to ˜59, 48, and 76%, respectively. Similar tofluorescence, the CD signal also indicated LEDGF₁₋₃₂₆ instability (FIG.15B).

On day 30 all formulations indicated significant difference in theellipticity at 208 nm as compared to day 0 signal. There was a hugebackground signal noise indicating presence of non-native structures.

LEDGF₁₋₃₂₆ indicated a mean hydrodynamic size of 7±1 nm one day 0 forall formulations accept sucrose containing formulation (FIG. 15C). Inpresence of sucrose LEDGF₁₋₃₂₆ indicated hydrodynamic size of 1 nm. Byday 30 particle size increased significantly to 578±366, 726±444,490±423, and 1052±125 nm, for plain buffer, Tween 20, EDTA, and sucrosecontaining formulations, respectively. For sodium azide group, insteadof increase in size there was decrease in size from 7 to 4 nm.

The monomeric native LEDGF₁₋₃₂₆ was monitored by SDS-PAGE (FIG. 16).Initially on day 0 the band intensity of LEDGF₁₋₃₂₆ at 40 kDa wassimilar in all groups. By day 7 there was thinning of 40 kda bandindicating loss of LEDGF₁₋₃₂₆ monomers. On day 14 appearance of lowermolecular weight bands in all groups were more pronounced as compared today 0.

Example 3 1. Materials and Methods

ARPE-19 cells were obtained from American Type Culture Collection (ATCC;Manassas, Va.). Cell culture materials, reagents and Lipofectamine 2000were obtained from Invitrogen Corporation (Carlsbad, Calif.). Chromicacid, HCl, NaOH and other supplies for circular dichroism were obtainedfrom Fisher Scientific (Pittsburgh, Pa.). Tris base, ZnCl₂, EDTA(ethylene diamine tera acetic acid) were obtained from Sigma-Aldrich(St. Louis, Mo.).

Preparation of Nanoassemblies

Lyophilized LEDGF₁₋₃₂₆ was dialyzed extensively overnight in 25 mMTris-HCl, 100 mM NaCl, pH 7.4 at 4° C. ZnCl₂ stock solution (100 mM) wasprepared in same buffer. For nanoassembly preparation, zinc stocksolution was diluted to final concentration of 0.1 mM, 1 mM, and 10 mMusing 25 mM Tris-HCl, 100 mM NaCl, pH 7.4 and LEDGF₁₋₃₂₆ (finalconcentration 1 mg/ml) was added to it and incubated at 37° C. for 24hr. LEDGF₁₋₃₂₆ with no zinc under similar conditions was kept ascontrol. All solutions were filtered with 0.2 μm filter beforepreparation of formulations. Nano (xx) assembly indicates LEDGF₁₋₃₂₆nanoassembly prepared with xx mM Zn(II).

Dynamic Light Scattering

Nanoassemblies homogeneity and size distribution was measured using zetasizer Nano ZS (Malvern, Westborough, Mass.) based on dynamic lightscattering (DLS). Briefly, sample was placed in 150 μl quartz cuvetteand data was collected at 173° backscatter angle with eleven scansaveraged for final size distribution plot. The time dependent variationin sizes of these nanoassemblies and their stability were monitored bymeasuring the number size distribution profile at different time points.

Fluorescence

The changes in the tertiary structure of LEDGF₁₋₃₂₆ were determined bymeasuring the steady state intrinsic fluorescence of tryptophan. Thesample was placed in 150 μl of quartz cuvette and emission spectra wererecorded from 300 to 400 nm, at 280 nm excitation wavelength, with anincrement of 1 nm using Spectramax M5 (Molecular Devices, Downingtown,Pa.). Number of scans per data point was 6.

Circular Dichroism

To determine the secondary structural changes in LEDGF₁₋₃₂₆, far-UV CDspectrum of the formulations were recorded. Briefly, sample was placedin 1 mm quartz cuvette and spectra was recorded at a scan speed of 0.5secs per time point, step size of 1 nm and the bandwidth of 4 nm from200 to 280 nm using Chirascan® CD instrument (Applied Photophysics Ltd,UK). All scans were done in triplicate. The scans were subtracted forbuffer component.

Cell Viability Assay

ARPE-19 cells were used to determine the cell survival function ofLEDGF₁₋₃₂₆ in presence of aggregation mediated stress. Briefly, ARPE-19cells were maintained as describer earlier (Baid et al. PLoS One.6(9):e24616). For cell viability assay, 10000 cells were seeded in96-well plate in serum containing DMEM-F12 media. After 24 hr, themedium was aspirated out and cells were washed with 100 μl of serum freemedium. Thereafter, cells were treated with 200 μl of either LEDGF₁₋₃₂₆alone or the LEDGF₁₋₃₂₆ +10 mM zinc nanoassemblies for 48 hr. No cells(just the media), cells with 25 mM Tris buffer as control for LEDGF₁₋₃₂₆alone, cells with 25 mM Tris +10 mM zinc (equivalent to group containinghighest amount of zinc) were maintained as control. The cells wereincubated for 24, and 48 hr. Thereafter, the medium was aspirated outand 200 μl of fresh serum free medium was added. 20 μl of MTT reagent(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide, 5 mg/mlin PBS pH 7.4) was added to each well and further incubation was donefor 3 hr at 37° C. The MTT containing medium was aspirated out and theformazan crystal formed was dissolved in 200 μl of DMSO. The absorbanceof the color developed was measured at 570 nm using Spectramax M5. Thepercentage viability of groups was calculated with reference to thecontrol group containing cells with no lipofectamine 2000. All groupswere repeated as n=3.

Cell Uptake

ARPE-19 (50,000) cells were seeded in 24-well plate in serum containingDMEM-F12 medium. After 24 hr the medium was aspirated out and cells werewashed with 100 μl of serum free medium. Thereafter, cells were treatedwith 200 μl of either LEDGF₁₋₃₂₆ or nano(10) assembly for 1 and 6 hr.Cells with 25 mM Tris buffer as control for LEDGF₁₋₃₂₆ alone, cells with25 mM Tris +10 mM zinc (equivalent to group containing highest amount ofzinc) were maintained as control. After 2 or 6 hr, cells were washedwith 500 μl of cold PBS pH 7.4 twice followed by 2 washes of 500 μl ofacid PBS pH 5. Thereafter, cells were lysed with 1% triton-x for 20 minat room temperature and scrapped and collected. Fluorescence wasmeasured at 488 nm excitation and 519 nm emission.

Chelation

For investigating whether the formation of nanoassemblies can bereversed in presence of chelating agent, the nanoassemblies were allowedto be formed for 24 hr at 37° C. as described above. EDTA 500 mM stocksolution was made using 25 mM Tris-HCl and 100 mM NaCl, pH 7.4. EDTA wasadded to final concentration of 50 mM to either LEDGF₁₋₃₂₆ ornanoassembly. It was incubated for 4 hr at room temperature, thereafterUV, fluorescence, and CD was taken as described above.

2. Results LEDGF₁₋₃₂₆ Undergoes Structural and Conformational Changes inPresence of ZnCl₂

Formation of nanoassemblies in presence of Zn(II) was monitored bydynamic light scattering (DLS), intrinsic trp-fluorescence, far-UV CD,and UV-vis spectra (FIG. 17). LEDGF₁₋₃₂₆ indicated a hydrodynamicdiameter of 9±1 nm, which did not change significantly within the periodof 24 hr as monitored by DLS (FIG. 17A). LEDGF₁₋₃₂₆ incubated withdifferent concentration of Zn(II) underwent a change in size, indicatingformation of nanoassemblies. With 0.1 mM ZnCl2 there no change in sizeuntil 24 hr. In presence of 1 mM and 10 mM Zn(II) LEDGF₁₋₃₂₆ indicatedan increase in size within 30 min of incubation at 37° C. At 24 hrincubation, LEDGF₁₋₃₂₆ indicated a size of 7±1, 22±5, 26±5, and 28±5 nmfor control, nano(0.1), nano(1), and nano(10) assemblies, respectively.

To investigate the changes in the secondary structure of LEDGF₁₋₃₂₆ inpresence of Zn(II), the far-UV CD spectra of the LEDGF₁₋₃₂₆ wasmonitored from 200-260 nm (FIG. 17B). After 24 hr incubation at 37° C.,LEDGF₁₋₃₂₆ indicated a negative ellipticity below 200 nm and a negativedip at 230 nm. Deconvolution of the spectra indicted that LEDGF₁₋₃₂₆ isprimarily a random coiled structure. In presence of Zn(II) significantchanges in CD spectra was indicated. The negative dip at 230 nm shiftedtowards left (to lower wavelength). The ellipticity of LEDGF₁₋₃₂₆indicated increased negative signal as the concentration of Zn(II)increased. The change in the CD was dependent on the Zn(II)concentration, being slow for 0.1 mM Zn(II) and fastest for 10 mM Zn(II)formulation (data not shown). There was a shift in the negative dip at230 nm towards lower wavelength in concentration dependent mannerindicating possible formation of α-helix. However below 200 nm thenegative signal also increased depending on the Zn(II) concentration.Deconvulation of CD spectra indicated 2% increase in the α-helix ascompared to control in nano (10) formulation.

The intrinsic trp fluorescence spectrum of LEDGF₁₋₃₂₆ (FIG. 17C)indicated in presence of Zn(II) the intensity of the fluorescence signaldecreased between 200-400 nm in presence of Zn(II). Interestingly thedecrease in fluorescence signal was more in 0.1 mM Zn(II) formulation ascompared to 10 mM Zn(II) formulation.

LEDGF₁₋₃₂₆ showed an A_(max) at 276 nm in UV absorbance spectra. (FIG.17D). In presence of Zn(II) there was a decrease in the UV signal for0.1 and 1 mm Zn(II) formulation but no change was observed for 10 mMzinc formulation. The UV signal at 276 nm (A_(max)) was 0.47, 0.33,0.32, and 0.46 for control, nano(0.1), nano(1), and nano(10) assemblies,respectively.

LEDGF₁₋₃₂₆ Forms Nanostructures

As shown in FIG. 18, LEDGF₁₋₃₂₆ nano (10) assemblies (right most panel)when visualized under transmission electron microscopy (TEM) indicatedpresence of loosely formed nanostructures as seen by dense negativestain. Some nanoassemblies clung to each other, while others werepresent as individual particle. In absence of Zn(II), LEDGF₁₋₃₂₆ (middlepanel) did not indicate presence of any such structures.

Nanoassemblies is Stable Over Dilutions

Stability of nanoassemblies over dilution was studied using DLS (FIG.10). Nano (10) assemblies once formed did not indicate any change insize when diluted 2 and 4 times. Further, when these diluted nano (10)assemblies were stored at 4° C. for 24 hr and then size taken there wasno change in size.

Formation of Nanoassembly is Reversible

To investigate the kind of interaction between LEDGF₁₋₃₂₆ and zinc, wemonitored the effect of EDTA on nano (10) (FIG. 19). Addition of EDTA tocontrol LEDGF₁₋₃₂₆ did not indicate any significant size change andLEDGF₁₋₃₂₆ size remained to ˜7-8 nm (FIG. 19A). Before addition of EDTA,nano (10) assembly indicated size of 25±4 which decreased to 9±2 uponEDTA addition. The fluorescence spectrum of nanoassembly which wasquenched due to formation of nanoassembly reverted back (FIG. 19B).After addition of EDTA, control and nano (10) assemblies indicatedsimilar fluorescence spectrum. All changes in the secondary structure asindicated by CD spectrum were similarly reverted (FIG. 19C). UV-visspectrum also indicated reversal of changes that occurred due toassembly formation (FIG. 19D).

LEDGF₁₋₃₂₆ Stability is Increased in Nanoassemblies

The stability of LEDGF₁₋₃₂₆ in nanoassemblies at 25° C. was monitoredfor 30 days using DLS, SDS-PAGE, CD, and fluorescence (FIG. 20). ControlLEDGF₁₋₃₂₆ size decreased from 8±1 to 5±1 nm on day 3 (FIG. 20A). Nano(1) assemblies indicated size of 27±1 at day 0 which decreased to 4±3 nmon day 7. On the other hand nano (10) assemblies indicated no change insize until day 30. On day 30 there was a size change from 27±1 to 8±2 nmfor nano (10) assemblies.

A 40 kDa band was indicated in SDS-PAGE in control LEDGF₁₋₃₂₆, nano (1)and nano (10) assemblies on day 0 (FIG. 20B). By day 3 there wascomplete loss of this band in control LEDGF₁₋₃₂₆. Nano (1) assemblieslost this band on day 7 while Nano (10) lost it on day 30.

The CD spectrum of control LEDGF₁₋₃₂₆ indicated a continuous loss innegative ellipticity from day 1 onwards and complete loss of signal onday 30 (FIG. 20C). Nano (1) assemblies indicated a continuous CD signalloss from day 1 onwards; however until day 30 there was CD signal. Therewas no significant signal loss in nano (10) assemblies until day 14, onday 30 there was decrease in CD signal, however the negative signal wasstill seen distinctively.

The fluorescence spectrum of control LEDGF₁₋₃₂₆ indicated significantdecrease in fluorescence intensity on day 7 as compared to day 0,further fluorescence loss continued day 30 (FIG. 20D). Nano (1)assemblies on the other hand indicated no loss in fluorescence signalsuntil day 7 as compared to day 0 signals, however on day 14 and onwardsthere was a signal loss. Nano (10) being most robust indicated no lossin intensity until day 14 as compared to day o signal and very lessdecrease in the intensity on day 30 as compared to other correspondinggroups.

ARPE-19 Cells Take Up Higher Amount of Nanoassemblies

The cellular uptake of LEDGF₁₋₃₂₆ was investigated in ARPE-19 cellsusing Alexa conjugated LEDGF₁₋₃₂₆ (FIG. 21). ARPE-19 uptake ofAlexa-LEDGF₁₋₃₂₆ was dose dependent. Within 2 hr, 0.5±0.1, 1.0±0.3, and1.4±0.2 μg of Alexa-LEDGF₁₋₃₂₆ (control) was taken up by ARPE-19 cellswhen incubated with 2, 10, and 25 μg/ml of Alexa-LEDGF₁₋₃₂₆,respectively. When incubation time was increased to 6 hr, there was anincrease in the uptake of Alexa-LEDGF₁₋₃₂₆. LEDGF₁₋₃₂₆ uptake increasedto 1.2±0.2, 1.2±0.2, and 1.9±0.2 μg in 6 hr for 2, 10, and 25 μg/ml ofAlexa-LEDGF₁₋₃₂₆ treatment, respectively.

Alexa-LEDGF₁₋₃₂₆ nano (10) assemblies indicated higher uptake at 2, and6 hr as compared to corresponding control groups. At 2 hr there was0.7±0.08, 1.0±0.2, and 2.2±0.5 μg uptake for 2, 10, and 25 μg/ml of nano(10) assembly treatment, respectively. Although nanoassembly uptake washigher as compared to control, there was no significant difference.Increasing the incubation time from 2 hr to 6 hr increased nano (10)assembly uptake significantly. The uptake of was 2.0±0.09, 2.9±0.5, and2.9±0.2 μg at 6 hr for 2, 10, and 25 μg/ml of nano (10) assemblytreatment, respectively.

Nanoassemblies Survives ARPE-19 Cells from Serum Starvation

Efficacy of nanoassemblies to survive ARPE-19 cells under serumstarvation was monitored by MTT assay. (FIG. 22). Compared to untreatedgroups (100% viability), LEDGF₁₋₃₂₆ treated group indicated an increasein viability in dose dependent manner. ARPE-19 cell viability increasedto 124±11, 148±12, and 160±44% with treatment of 0.01, 0.1, and 1.0μg/ml of LEDGF₁₋₃₂₆ treatment, respectively. Nano (10) assembliestreated group indicated higher cell viability compared to correspondingcontrol LEDGF₁₋₃₂₆ treated groups. ARPE-19 cell viability increased to150±3, 180±22, and 200±8% with treatment of 0.01, 0.1, and 1.0 μg/ml ofLEDGF₁₋₃₂₆ treatment, respectively.

Nanoassembly Increases LEDGF₁₋₃₂₆ Efficacy in Delaying RetinalDegeneration

Efficacy of nanoassemblies to prevent the functional loss in retina wastested in RCS rats using electroretinography (ERG) (FIG. 23A). On week4, before the intravitreal injection was given to rats, the b-waveamplitude was 313±32 μV, with no significant difference in all groups.Over a period of subsequent 10 weeks i.e. at 14 week the buffer andZn(II) treated group indicated a decrease in b-wave amplitude from307±44 to 17±10 and 302±37 to 11±7 μV, respectively. LEDGF₁₋₃₂₆treatment (control) slowed the loss in the b-wave amplitude and on14^(th) week the b-wave amplitude declined from 337±30 to 42±15 μV,however, there was no significant difference in the buffer, Zn(II) and,control group b-wave amplitude in week 14. Nano (10) indicated asignificant protection against the loss of b wave amplitude until 14week. The b-wave amplitude declined from 305±36 to 65±15 μV on week 14indicating significantly high b-wave as compared corresponding buffer orZn(II) group.

On week 4 the b-wave amplitude of all groups was 105±23 in photopic ERG(FIG. 23B). Similar to scotopic ERG, a decrease in b-wave amplitude wasseen. The buffer and Zn(II) treated groups indicated a loss in b-waveamplitude from 94±26 to 12±7 and 109±23 to 11±7 μV, respectively.Control LEDGF₁₋₃₂₆ and nano (10) assemblies treated group delayed theloss, and the b-wave amplitude decreased from 100±28 to 22±5 and 113±23to 40±10 μV, respectively. Nano (10) assemblies indicated significantlyhigher b-wave as compared to corresponding buffer, Zn(II) and controlLEDGF₁₋₃₂₆ treated group.

Further oscillatory potential (OP) amplitude in all groups was 91.8±11.5μV at week 4. (FIG. 23C). There was decrease in (OP) amplitude was seenacross all groups. By week 14 the OP amplitude was 32±5, 33±8, 36±8, and36±9 μV, in buffer, Zn(II), control, and nano (10) assembly group,respectively. There was no significant difference in all groups.

The 30 hz flicker amplitude was also measured. (FIG. 23D). On week 4 theflicker amplitude was on an average 10±2 μV for all groups. As with anyother ERGs there was decease in the flicker amplitude in all groups;however on week 14 the flicker amplitude of nano (10) assembly group wassignificantly higher than buffer, and Zn(II) group. The flickeramplitude values on week 14 was 2±0.4, 3.7±0.4, 2.4±0.6, 5.2±2.2 μV forbuffer, Zn(II), control and nano (10) assemblies, respectively.

Nanoassembly Increases LEDGF₁₋₃₂₆ Persistence for Days in Ocular Tissues

Persistence of LEDGF₁₋₃₂₆ in normal SD rat was measured using thefluorescence signal of Alexa-LEDGF₁₋₃₂₆ (FIG. 24). FIG. 24A indicates anaverage of blank scan (n=7) before the intravitreal injection of the SDrat eye. The blank scan indicated autofluorescence of choroid, lens, andcornea at about 24, 50, and 88 data points, respectively. Based on thisblank scan data points were assigned to various tissues of the eye. Thedata points assigned were—Choroid-RPE-24, vitreous humor ˜40, lens-50,aqueous humor ˜70, and cornea-88. FIGS. 24B and 32C is the standardcurve for control and nano(10) assembly. This was used to convert thefluorescence signal obtained in term of sodium fluorescein (NaF)concentration (ng/ml) from the Flurotron scans to actualAlexa-LEDGF₁₋₃₂₆ concentration (μg/ml). FIGS. 24D and 24E are Flurotronscans (N=4) for control and nano (10) assembly groups from 2 min to 14days after intravitreal injection. The fluorescence signal in vitreous,choroid-RPE, and aqueous humor was obtained from the Flurotron scans(FIGS. 24D, and 24E) and was converted to actual Alexa-LEDGF₁₋₃₂₆concentration in FIGS. 24F, 24G, and 24H, respectively. A high peak inthe vitreous at 2 min of injection in FIGS. 24C, and 24D indicatedintravitreal injection was rightly done.

A fluorescence signal equivalent to 3±0.5 ng/ml of NaF was observed invitreous in blank scans (FIG. 24A) which when converted resulted in 0μg/ml of Alexa-LEDGF₁₋₃₂₆ as base line. After 2 min of the intravitrealinjection, a peak value of 292±106 μg/ml of Alexa-LEDGF₁₋₃₂₆ wasindicated in the vitreous for control, which sharply declined to 127±74μg/ml in 30 min (FIG. 24F). By day 3 the Alexa-LEDGF₁₋₃₂₆ peak incontrol groups was below the base line. Nano (10) assembly on the otherhand indicated days of persistence. The peak concentration after 2 minof injection was 321±54 μg/ml of Alexa-LEDGF₁₋₃₂₆, there was initialrapid decline to 100±45 μg/ml in 30 min; thereafter the decline was slowas compared to control and persistence of nanoassembly in vitreous wasindicated until day 14. On day 14 Alexa-LEDGF₁₋₃₂₆ concentration was0.7±0.1 μg/ml in the nano (10) assembly group which was significantlyhigher than control group and the base line.

Persistence of Alexa-LEDGF₁₋₃₂₆ was also indicated in the choroid-RPE aswell as in aqueous humor for the nano (10) assembly group. Theautofluorescence in the choroid RPE indicated 10±4 ng/ml of NaF, whichwhen converted to Alexa-LEDGF₁₋₃₂₆ concentration was 0.1 μg/ml. Thus 0.1μg/ml of Alexa-LEDGF₁₋₃₂₆ was considered as base line for Choroid-RPE(FIG. 24G). Soon after intravitreal injection 13.2±10.8 μg/ml ofALexa-LEDGF₁₋₃₂₆ was indicated in choroid-RPE which increased to30.0±22.6 μg/ml in 30 min in control group. Thereafter, there wasdecline in Alexa-LEDGF1-326 level and was undetectable by day 1. Nano(10) assembly on the other hand indicated 14.3±9.8 μg/ml ofAlexa-LEDGF₁₋₃₂₆ in 2 min which increased to 21.5±12.8 μg/ml in 30 min.By day 1 Alexa-LEDGF₁₋₃₂₆ level dropped to 2.0±1.1 μg/ml, however it wassignificantly high from the base line an control group. Alexa-LEDGF₁₋₃₂₆level remained significantly high until day 14 and was detected to be0.6±0.2 μg/ml on day 14.

In aqueous humor the base line calculated from the blank scan wasequivalent to 0 μg/ml of Alexa-LEDGF₁₋₃₂₆ (FIG. 24A). At 2 min afterintravitreal injection, Alexa-LEDGF₁₋₃₂₆ concentration was 3.4±3 and4.9±2.4 μg/ml for control, and nano (10) assembly group (FIG. 24H).Alexa-LEDGF₁₋₃₂₆ dropped below the base line within 6 hr for the controlgroup, while the nano (10) assembly indicated presence ofAlexa-LEDGF₁₋₃₂₆ until day 14, at this time point the concentration ofAlexa-LEDGF₁₋₃₂₆ was 0.6±0.1 μg/ml.

LEDGF₁₋₃₂₆ Remains Immune Reactive In Vivo for Days when Dosed as aNanoassembly

Alexa-LEDGF₁₋₃₂₆ immunoreactivity was investigated in various oculartissues and in blood after 14 days of single intravitreal injection byindirect ELISA (FIG. 25). There was no detectable level ofAlexa-LEDGF₁₋₃₂₆ in the cornea, aqueous humor, lens, vitreous humor,retina, choroid-RPE, sclera and blood for the un-injected blank eyetissues indicating absence of any detectable quantity of endogenousLEDGF₁₋₃₂₆. Interestingly, Alexa-LEDGF₁₋₃₂₆ was detectable in retina forboth control and nano(10) assembly group being 0.2±0.2 and 1.3±0.4 μg/gof tissue weight, respectively. There was no significant difference inthe control and blank group, while nano(10) assembly had significantlyhigher amount of Alexa-LEDGF₁₋₃₂₆ as compared to control or blank group.

Example 4

Live/Dead Cell Count Assay—

For cell count assay, 10000 ARPE-19 cells were plated in 96-well plateand incubated for 24 hours. (FIG. 26). After 24 hours, the serumcontaining medium was aspirated out. The test groups (pP23H-Rho+LEDGF₁₋₃₂₆) were transiently transfected with pP23H-Rho plasmid (1μg/ml) using 1:3 ratio of lipofectamine 2000 (LP-2000) in serum freemedium as per manufacturer's protocol. After six hours of transfection,the medium were aspirated out and cells were treated with increasingamount of LEDGF₁₋₃₂₆. No cells (just the medium), cells with no LP-2000and cells with LP-2000 were also maintained as control. At the end ofLEDGF₁₋₃₂₆ treatment period, cells were washed with PBS. The cells werelabeled with a combination of plasma membrane permeant (Hoechst 33342),a plasma membrane impermeanble molecule (BOBO™ 3), and a nuclear dye(4′,6-diamidino-2-phenylindole, dihydrochloride; DAPI). Hoechst 33342labeled cell nuclei, whereas BOBO™ 3 labeled dying or dead cells. Thecells were visualized using Operetta® high content imaging system. Cellcount was obtained using automated software tool in the Operetta®instrument. Number and percentage of live cells were calculated bysubtracting the dead cell count from “all cell” count. As shown in FIG.26, LEDGF₁₋₃₂₆ increases ARPE-19 cell viability.

Imunoblotting:

For immunoblotting CFP tagged P23H-Rho (pP23H-CFP-Rho) was used insteadof P23H-Rho for transfection. ARPE-19 cells were plated in 60 mm plates;transfection and drug treatment was up scaled proportionately relativeto 96-well plate study. After LEDGF₁₋₃₂₆ treatment is over, cells werewashed once with cold PBS and lysed by sonication in RIPA buffercontaining protease inhibitor. Equal amount of supernatant were loadedinto SDS-PAGE gel and was immunoblotted for Hsp70, Hsp40, Hsp27, CFP(for P23H-CFP-Rho), and LEDGF₁₋₃₂₆, and β-actin. Protein bands werevisualized using enhanced chemiluminecence ECL™ detection kit (GEHealthcare, Piscataway, N.J.). The observed data indicate thatLEDGF₁₋₃₂₆ is internalized

Phagocytic Assay—

ARPE-19 cells were seeded in 24-well plates and transfected with 20pM/ml of MERTK siRNA (Santa Cruz Biotechnology Inc., Dallas, Tex.),using siRNAtransfecting agent (Santa Cruz Biotechnology Inc., Dallas,Tex.) for 6 hours. The transfecting medium was removed and cells werefurther incubated in serum free medium for 24 hours. Cells transfectedonly with the transfecting medium and no MERTK siRNA were maintained ascontrol. Cells were washed once and treated with 0.05, 0.5, or 5 μg/mlof LEDGF₁₋₃₂₆ for 24 hours and then phagocytosis of 2 μm particles weremonitored. Briefly, 100 μg/ml of 2 μm blue FluoSpheres (LifeTechnologies, Grand Island, N.Y.) was incubated with cells for 3 hours.Thereafter, cells were washed twice with cold PBS pH 7.4, followed bytwo washes of cold PBS pH 5.0 to remove adherent FluoSpheres. Cells werelysed using 1% Triton-x, and the fluorescence of the particles in thecell lysate was measured using 350 nm excitation and 430 nm emission.Cells transfected with only transfecting agent without siRNA was takenas control for particle uptake. Cells with no particle treatment wereused for background fluorescence measurements. As shown in FIG. 27,LEDGF₁₋₃₂₆ increases phagocytic activity. Decreased phagocytosis ofretinal pigment epithelial cells is a hallmark of several retinaldiseases including degenerative diseases. LEDGF₁₋₃₂₆ will be useful intreating diseases with impaired phagocytosis.

Histology:

At the end of the study i.e. on 12^(th) week, eyes were enucleated afterERG measurements and fixed in Davidson's fixative (2% of 37-40%formaldehyde, 35% ethanol, 10% glacial acetic acid, and 53% distilledwater) for 24 hours at room temperature. The eyes were then stored in70% ethanol for subsequent serial dehydration and embedment in paraffin.Three vertical sections of 6 lam thick were cut from the nasal to thetemporal side at the optical nerve (500 μm apart) on a standardmicrotome. Gross retinal morphology was assessed by light microscopefollowing hematoxylin/eosin staining of tissue sections. The thicknessof outer nuclear layer (ONL) and inner nuclear layer (INL) was measuredmethodically using Aperio ImageScope software v11.1.2.760. Since thephotoreceptor cell protection may be uneven across the retina, every 500μm from the superior edge to the inferior edge in each section wasanalyzed and average of three sections was done for each point. Datarepresent average of three eyes. As shown in FIG. 28, LEDGF₁₋₃₂₆ delaysboth retinal nuclear photoreceptors loss.

Immunofluorescence:

For immunofluorescence, after removal of paraffin, eye sections wereprocessed through the following sequential steps at room temperature,unless otherwise indicated. Antigen was retrieved by boiling thesections at 80° C. for 15 min. After blocking the nonspecific binding,sections were incubated with mouse anti-rhodopsin (1D4) primary antibodyat 4° C. overnight followed by 30 min incubation with Alexa Fluor® 594conjugated donkey anti-mouse IgG and DAPI. Finally, eye sections werewashed and mounted by Supermount H (Biogenex, San Ramon, Calif.)mounting medium to prevent rapid loss of fluorescence. The fluorescencewas visualized using confocal microscope (Nikon Eclipse C1) at 20×optical zoom. The excitation-emission wavelengths used for DAPI and,Alexa Fluor were 408-450/35, and 637-605/75 nm, respectively. Imageswere captured using Nikon EZ-C1 software version 3.40. As show in FIG.29, LEDGF₁₋₃₂₆ delays rod outer segment loss.

Example 5 In Vitro Cumulative Release of His-LEDGF₁₋₃₂₆

His-LEDGF₁₋₃₂₆ encapsulated NPinPMP were evaluated for in vitro releasein PBS pH 7.4. Particles (2-3 mg) were weighed and dispersed in 1 ml ofPBS pH 7.4 and incubated at 37° C. under shaking at 200 rpm (Max Qshaker incubator). At predetermined time points the suspended particleswere centrifuged at 13,000 g for 15 min and the supernatant wascollected. The pellet comprising particles was resuspended in 1 ml offresh PBS pH 7.4 and incubated. The His-LEDGF₁₋₃₂₆ content in thesamples was estimated using micro BCA assay as per the manufacturer'sinstructions (Pierce Biotechnology, IL, USA). The in vitro cumulativedata showed the sustained release of His-LEDGF₁₋₃₂₆ from NPinPMP. Asshown in FIG. 30, a cumulative 60% release of His-LEDGF₁₋₃₂₆ wasobserved by the end of 3 months.

In Vivo Delivery of his-LEDGF₁₋₃₂₆ in Rats

In vivo delivery of His-LEDGF₁₋₃₂₆ was evaluated following intravitrealadministration of Alexa Fluor 488 conjugated His-LEDGF₁₋₃₂₆ in NPinPMPin a rat model. No unlabeled LEDGF₁₋₃₂₆ was used in the NPinPMP. The rateyes were injected with Alexa-His-LEDGF₁₋₃₂₆ encapsulated NPinPMP (6.0μg of His-LEDGF₁₋₃₂₆/5 μl) and as a control Alexa-His-LEDGF₁₋₃₂₆=atequivalent concentration (1.5 μg labeled protein and 4.5 μg unlabeledprotein/5 μl) was injected. This ratio allowed us to start with asimilar fluorescence intensity for both groups to begin with. Ocularfluorescence due to the release of Alexa-His-LEDGF₁₋₃₂₆ was monitoredperiodically using Fluorotron Master™ (Ocumetrics, CA, USA) until thefluorescence reached the lower detection limit or baseline. Baselinefluorescence values of eyes were monitored before injecting theformulations. At each time point, three fluorometric scans were takenand mean value was used. Standard curve for Alexa-His-LEDGF₁₋₃₂₆ atdifferent concentrations was obtained using a cuvette and ocularflurophotometry with a rat lens adapter. The standard curve was used toconvert fluorescein equivalent concentrations provided byfluorophotometer to actual Alexa-His-LEDGF₁₋₃₂₆ concentration.

After intravitreal injection of Alexa-His-LEDGF₁₋₃₂₆ encapsulatingNPinPMP, and soluble Alexa-His-LEDGF₁₋₃₂₆, the concentrationsdistribution of His-LEDGF₁₋₃₂₆ along the eye optical axis was determinedindirectly by measuring the alexa fluorescence intensity distribution(equivalent of sodium fluoresciene concentration) curve along axialplanes, indicated as data points in an anterior to posterior direction.The fluorescence scans revealed sustained delivery ofAlexa-His-LEDGF₁₋₃₂₆ from NPinPMP compared to solution. Fluoresceinequivalent concentrations reported by Fluorotron Master were convertedto Alexa-His-LEDGF₁₋₃₂₆ concentrations. The Alexa-His-LEDGF₁₋₃₂₆concentration in the vitreous region from solution and NPinPMP group atdifferent time points was plotted. Only the concentrations of thelabeled bevacizumab are reported. Before intravitreal injection, thebaseline fluorescence readings of normal eyes were taken and thebaseline fluorescence concentration was found to be 2.03 μg/ml. As showin FIG. 31, the Alexa-His-LEDGF₁₋₃₂₆ solution injected group showedAlexa-His-LEDGF₁₋₃₂₆ concentration of 2.02 μg/ml on day 1 indicatingrapid elimination from vitreous region. In NPinPMP injected group theAlexa-His-LEDGF₁₋₃₂₆ the initial concentration in the vitreous was foundto be 18.23 μg/ml and the Alexa-His-LEDGF₁₋₃₂₆ concentration above thebaseline was maintained until 35th day and reached normal base linelevels by end of 50 days. The observed data indicate the ability toachieve sustained in vivo release of Alexa-His-LEDGF₁₋₃₂₆ from anexemplary PinP composition.

1. A peptide comprising an amino acid sequence of lens epitheliumderived growth factor (LEDGF), wherein the peptide comprises a LEDGFN-terminal stress related binding domain.
 2. The peptide of claim 1,wherein the peptide further comprises a TAT binding domain.
 3. Thepeptide of claim 1, wherein the peptide has a molecular weight ofapproximately 40 kDa.
 4. The peptide of claim 1, wherein the peptide hasan amino acid sequence of SEQ ID NO: 2., or an amino acid sequence withat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% sequence identity to SEQ ID NO:
 2. 5-6. (canceled)
 7. Acomposition comprising the peptide of claim
 1. 8. The composition ofclaim 7, further comprising a pharmaceutical carrier, diluent, excipientor combination thereof.
 9. The composition of claim 7, wherein thepeptide is bound to or associated with a colloidal metal nanoparticle.10-12. (canceled)
 13. The composition of claim 7, wherein the peptide isbound to, or encapsulated in, an inner particle, and wherein the innerparticle is loaded in a porous outer particle.
 14. The composition ofclaim 13, wherein the inner particle is made from a particle materialthat does not expand in supercritical carbon dioxide.
 15. Thecomposition of claim 13, wherein the inner particle material is apolymeric material. 16-17. (canceled)
 18. The composition of claim 13,wherein the outer particles are made from a particle material thatexpands in supercritical carbon dioxide.
 19. The composition of claim18, wherein the outer particle is a polymeric material. 20-21.(canceled)
 22. A method of treating a protein aggregation-mediateddisease comprising administering to a patient in need thereof acomposition according to claim
 7. 23. The method of claim 22, whereinthe aggregation-mediated disease is an ocular disease or aneurodegenerative disease.
 24. The method of claim 23, wherein theocular disease is retinal degeneration disease.
 25. The method of claim24, wherein the retinal degeneration disease is age related maculardegeneration (AMD), or retinitis pigmentosa (RP).
 26. (canceled)
 27. Themethod of claim 23, wherein the neurodegenerative disease is Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),amyotrophic lateral sclerosis, or a prion disease.
 28. A method ofreducing protein aggregation in a protein aggregation-mediated diseasecomprising administering to a patient in need thereof a compositionaccording to claim
 7. 29. The method of claim 28, wherein proteinaggregation is caused by endoplasmic reticulum stress, oxidative stress,or both.
 30. The method of claim 28, wherein the patient has retinaldegeneration disease or a neurogenerative disease. 31-33. (canceled)